Anti-ryk antibodies and methods of using the same

ABSTRACT

Methods for inhibiting degeneration of a neuron, methods of treating a neurological/neurodegenerative disease, methods of modulating the directional growth of a neuron, and methods of interfering with the interaction of Wnt and Ryk are provided herein. Also provided are isolated anti-Ryk antibodies and antibody fragments that specifically bind to a binding domain of Wnt.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of priority under 35 U.S.C. § 119(e)of U.S. Ser. No. 62/314,025, filed Mar. 28, 2016, the entire content ofwhich is incorporated herein by reference.

GRANT INFORMATION

This invention was made with government support under Grant Nos.NS047484 and NS081738 awarded by the National Institutes of Health. TheUnited States government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 15, 2017, isnamed 20378-201389_SL.txt and is 14,104 bytes in size.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates generally to antibodies and more specifically touse of an anti-Ryk antibody or antibody fragment that specifically bindsto a binding domain of Wnt to inhibit Wnt-Ryk signaling.

Background Information

The central nervous system (CNS) is connected by ascending sensorypathways and descending motor or regulatory pathways. In the CNS,somatosensory pathways ascend to the brain centers, and motor pathwayscontrolling body movement descend from the brain to the spinal cord.Unlike the peripheral nervous system, damage to the central nervoussystem axons, such as spinal cord axons cannot be repaired, causingpermanent impairment of neural function, such as in paralysis. Thespinal cord serves important functions in the central nervous system.One such function is to allow communication of the body and the brain.The nerve fibers within the spinal cord carry messages to and from thebrain to other parts of the body. In general, sensory information fromthe body travels along the spinal cord up to the brain and instructionfrom the brain, such as motor command, travels along the spinal corddown from the brain.

The term “spinal cord injury” refers to any injury of the neurons withinthe spinal canal. Spinal cord injury can occur from either trauma ordisease to the vertebral column or the spinal cord itself. Most spinalcord injuries are the result of trauma to the vertebral column causing afracture of the bone, or tearing of the ligaments with displacement ofthe bony column producing a pinching of the spinal cord. The majority ofbroken necks and broken backs, or vertebral fractures, do not cause anyspinal cord damage; however, in 10-14% of the cases where a vertebraltrauma has occurred, the damage is of such severity it results in damageto the spinal cord. It is estimated that the annual incidence of spinalcord injury (SCI), not including those who die at the scene of theaccident, is approximately 40 cases per million population in the U.S.,or approximately 11,000 new cases each year. The number of people in theU.S. who are alive today and who have SCI has been estimated to bebetween 721 and 906 per million population. This corresponds to between183,000 and 230,000 persons. Treatment options for patients with spinalcord injuries are limited. Often, patients with SCI are left withsevere, permanent disabilities.

In recent years, increasing evidence suggests that Wnts, which have beenbetter known as morphogens in early development, are conserved axonguidance molecules during nervous system wiring both in vertebrates andinvertebrates (Zou, Y. 2004. Trends Neurosci 27:528-32; Fradkin, et al.2005. J Neurosci 25:10376-8; Zou & Lyuksyutova. 2007. Curr OpinNeurobiol 17:22-8; Salinas, et al. 2008. Annu Rev Neurosci 31:339-358).Wnts are secreted glycoproteins, which bind to three classes ofreceptors, the Frizzleds, Ryk and ROR2 (Gordon & Nusse. 2006. J BiolChem 281:22429-33; Logan & Nusse. 2004. Annu Rev Cell Dev Biol20:781-810). It has also been shown that the Wnt family proteins areessential guidance cues along the A-P axis of the spinal cord andtopographic map formation in the retinotectal projections in developmentand may play important roles in regulating adult CNS axon regenerationafter spinal cord injury (Lyuksyutova et al. 2003. Science 302:1984-8;Liu, et al. 2005. Nat Neurosci 8:1151-9; Schmitt et al. 2006. Nature439:31-7; Wolf et al. 2008. J Neurosci 28:3456-67; Liu et al. 2008. TheJournal of Neuroscience 28:8376-8382).

Commissural axons, which originate from the dorsal spinal cord, firstproject along the dorsal-ventral axis to grow towards the ventralmidline, the floor plate. The ventrally directed growth of commissuralaxons are guided by repulsive cues, the BMPs emanating from the dorsalmidline, the roof plate, and attractive cues, Netrin-1 and SonicHedgehog, secreted from the floor plate (Zou et al. 2007. Curr OpinNeurobiol 17:22-8). Once these commissural axons cross the midline, theylose responsiveness to midline attractants and gain sensitivity tochemorepellents, the Slits and Semaphorins, emanating from the floorplate and the neighboring ventral gray matter, forcing them to make a90° turn into their longitudinal trajectory (Zou et al. 2000. Cell102:363-75). The dorsal populations of rodent commissural axons all turnanterior and project towards the brain. The anterior turning requiresWnt-Frizzled signaling. Several Wnts, including Wnt4, Wnt7b, Wnt7a andWnt5a, are expressed in an anterior-posterior decreasing gradient alongthe spinal cord at the ventral midline and attract post-crossingcommissural axons, which have crossed the midline to turn anteriorly.When the Wnt gradient was disrupted by adding Wnt inhibitors, secretedFrizzled-related proteins (sFRPs) or positioning Wnt4-secreting cellaggregates in “open-book” explant culture, commissural axons showspecific A-P randomize growth after midline crossing. In Frizzled3mutant embryos, spinal cord commissural axons lose A-P directionality invivo (Lyuksyutova et al. 2003. Science 302:1984-8).

Studies in Drosophila midline axon pathfinding independently showed thatDWnt5 is a chemorepellent and repels a subset of commissural axons via areceptor called, Derailed (Yoshikawa et al. 2003. Nature 422:583-8). Thevertebrate homologue of Derailed, Ryk, is also a repulsive Wnt receptorand an anterior-high posterior-low Wnt gradient created by differentialexpression of Wnt1 and Wnt5a, is required for the posterior growth ofcorticospinal tract axons in the spinal cord (Liu et al. 2005. NatNeurosci 8:1151-9). Therefore, Wnts control A-P guidance of bothascending and descending axons in the spinal cord by attractive andrepulsive guidance mechanisms. Wnt-Ryk signaling was also found toregulate the pathfinding of corpus callosum in the mammalian forebrainby a repulsive mechanism (Keeble et al. 2006. J Neurosci 26:5840-8).Studies in C. elegans showed that Wnt signaling controlanterior-posterior directionality of the pathfinding of a number ofaxons and migration of neuroblasts (Pan et al. 2006. Dev Cell 10:367-77;Hilliard et al. 2006. Dev Cell 10:379-90; Prasad & Clark. 2006.Development 133:1757-66). Therefore, the A-P guidance mechanisms appearto be highly conserved in animal kingdom (Zou. 2006. Neuron 49:787-9.).

In addition to the role of Wnts in axon pathfinding, Wnt3 is also apositional cue for topographic mapping in the retinotectal system,acting as a laterally-directing mapping force for retinal ganglion cellaxons, opposing the medially-directed force created by ephrinB1 gradientin the optic tectum (Schmitt et al. 2006. Nature 439:31-7). Wnt3 isexpressed in a medial-high to lateral-low gradient in the optic tectum.Ryk is expressed in a dorsal-ventral (D-V) increasing gradient in theretinal ganglion cells. Therefore, the more ventral RGC axon branchesare more strongly repelled by Wnt3 and Wnt3-Ryk signaling drives theinterstitial branches to grow towards the lateral tectum. In themeantime, ephrinB1 is expressed in the same graded fashion and EphBs areexpressed at higher levels in ventral RGCs. EphBs mediate attraction toephrinB1. Therefore, the more ventral RGC axon branches are moreattracted by ephinB1 towards the medial tectum. The balancing actbetween the medial (ephrinB1) and lateral (Wnt3) mapping forces ensuresRGC axons to terminate at correct topographic positions. Remarkably,Wnt-Frizzled signaling is also required for proper dorsal-ventralretinotopic mapping in the Drosophila visual system (Sato et al. 2006.Nat Neurosci 9:67-75; Zou & Lyuksyutova. 2007. Curr Opin Neurobiol17:22-8). Therefore, Wnts are conserved topographic mapping cues alongthe D-V axis.

Commissural axons of the developing spinal cord are guided to theventral midline by a collaboration of chemoattractants (Netrin-1 andSonic Hedgehog (Shh)) and chemorepellents (Bone Morphogenetic Proteins(BMPs)) secreted by midline floor plate and roof plate cells,respectively. Once these axons reach the floor plate they switch offtheir responsiveness to chemoattractants from the floor plate and becomeresponsive to chemorepulsive cues also expressed by the floor platecells and the surrounding ventral gray matter, including members of theClass 3 Semaphorins (Sema3B and Sema3F) and the Slit family proteins(Serafini, T., et al. Cell 78, 409-424, 1994; Kennedy, T. E., et al.Cell 78, 425-435, 1994; Serafini, T., et al. Cell 87, 1001-1014, 1996;Zou, Y., et al. Cell 102, 363-375, 2000; Charron, F., et al. Cell 113,11-23, 2003; Long, H., et al. Neuron 42, 213-223, 2004). Neuropilin-2mutant embryos showed severe guidance defects including stalling in themidline, overshooting to the contralateral side of the spinal cord andrandomly projecting along the anterior-posterior axis (Zou, Y., et al.Cell 102, 363-375, 2000).

The identification of modulators of neuronal growth and regenerationfollowing SCI could be applied in new forms of treatment of patientswith this debilitating condition. The identification of modulators ofneuronal growth and regeneration could also be applied in the treatmentof patients with other disorders involving neuronal dysfunction, such asneurological/neurodegenerative diseases or disorders. Agents that canpromote axonal growth along the A-P axis following injury to the spinalcord may be applied to help prevent the permanent paralysis that isoften associated with SCI. Therefore, there is a need for bettertreatments of SCI, and a greater understanding of modulators of neuronalgrowth and regeneration might lead to improved methods of treatment ofsuch neurological/neurodegenerative diseases or disorders.

SUMMARY OF THE INVENTION

The present invention is based on the finding that an anti-Ryk antibodyor antibody fragment that specifically binds to a binding domain of Wntinhibits Wnt-Ryk signaling. As such, the anti-Ryk antibody or antibodyfragment may be used to modulate the directional growth of a mammalianneuron when the spinal cord has been damaged, as well as to inhibitdegeneration of a neuron and to treat a neurodegenerative disease.

Accordingly, in one aspect, the invention provides an isolated anti-Rykantibody or antibody fragment that specifically binds to a bindingdomain of Wnt or specifically binds to the same epitope on Wnt as does areference antibody or antibody fragment, or cross-competes for specificbinding to Wnt with a reference antibody or antibody fragment. Invarious embodiments, the antibody or antibody fragment and/or thereference antibody or antibody fragment includes a heavy chain variableregion comprising the CDR sequences set forth in SEQ ID NOs: 5-7 or SEQID NOs: 5, 11, and 12; and/or a light chain variable region comprisingthe CDR sequences set forth in SEQ ID NOs: 1-3 or SEQ ID NOs: 9, 2, and3. In various embodiments, the antibody or antibody fragmentspecifically binds to an epitope within amino acid residues 90-183 ofWnt. In various embodiments, the heavy chain variable region of theantibody or antibody fragment includes an amino acid sequence comprisingat least 85% sequence identity, at least 90% sequence identity, at least95% sequence identity, or at least 99% sequence identity to SEQ ID NOs:8 or 13. In various embodiments, the light chain variable region of theantibody or antibody fragment includes an amino acid sequence comprisingat least 85% sequence identity, at least 90% sequence identity, at least95% sequence identity, or at least 99% sequence identity to SEQ ID NOs:4 or 10. In various embodiments, the antibody or antibody fragment isformulated in a pharmaceutically acceptable carrier or excipient.

In another aspect, the invention provides a nucleic acid sequenceencoding the isolated antibody or antibody fragment described herein.Also provided is a vector, such as an expression vector, that includesthe nucleic acid sequence. Also provided is a host cell, such as amammalian host cell that includes the vector.

In another aspect, the invention provides an immunoconjugate of theisolated anti-Ryk antibody or antibody fragment linked to a therapeuticagent such as a cytotoxin or a radioactive isotope. In another aspect,the invention provides a bispecific molecule, e.g., a bispecificantibody, which includes the isolated anti-Ryk antibody or antibodyfragment linked to a second functional moiety having a different bindingspecificity than the isolated anti-Ryk antibody or antibody fragment.

In another aspect, the invention provides a method of interfering withinteraction of Wnt and Ryk. The method includes contacting a sample thatincludes Wnt and Ryk with the isolated antibody or antibody fragmentdescribed herein, thereby interfering with the interaction of Wnt andRyk.

In another aspect, the invention provides a method of inhibitingdegeneration of a neuron. The method includes contacting the neuron withthe isolated antibody or antibody fragment described herein, therebyinhibiting degeneration of the neuron. In various embodiments,degeneration of an axon of the neuron is inhibited or degeneration of acell body of the neuron is inhibited. In various embodiments, the axonis a spinal cord commissural axon, an upper motor neuron axon, or acentral nervous system axon. In various embodiments, the neuron is adamaged spinal cord neuron, a sensory neuron, a motor neuron, acerebellar granule neuron, a dorsal root ganglion neuron, a corticalneuron, a sympathetic neuron, or a hippocampal neuron. In variousembodiments, the neuron forms part of a nerve graft or a nervetransplant. In various embodiments, the neuron is ex vivo or in vitro.In various embodiments, the nerve graft or the nerve transplant formspart of an organism, such as a mammal or human.

In another aspect, the invention provides a method of treating aneurological disease or disorder, e.g., a neurodegenerative disease ordisorder in a subject having or being at risk of developing theneurological disease or disorder, e.g., a neurodegenerative disease ordisorder. The method includes administering to the subject the isolatedantibody or antibody fragment described herein, thereby treating theneurological disease or disorder, e.g., a neurodegenerative disease ordisorder, in the subject. In various embodiments, the neurodegenerativedisease is amyotrophic lateral sclerosis, Alzheimer's disease orParkinson's disease.

In another aspect, the invention provides a method for modulating thedirectional growth of a mammalian neuron. The method includes contactingthe neuron with the isolated antibody or antibody fragment describedherein, thereby modulating the directional growth of the neuron, aspinal cord commissural axon, an upper motor neuron axon, or a centralnervous system axon. In various embodiments, the neuron is a damagedspinal cord neuron, a sensory neuron, a motor neuron, a cerebellargranule neuron, a dorsal root ganglion neuron, a cortical neuron, asympathetic neuron, or a hippocampal neuron. In various embodiments, theneuron forms part of a nerve graft or a nerve transplant. In variousembodiments, the neuron is ex vivo or in vitro. In various embodiments,the directional growth of the neuron facilitates regeneration of theneuron.

In another aspect, the invention provides a transgenic non-human mammalsuch as a mouse whose genome comprises a heterozygous or homozygousdeletion, inactivation or knock-out of the Ryk gene. In variousembodiments, the mouse has the phenotype Frizzled3^(−/−) Ryk^(+/−). Invarious embodiments, the mouse contains a conditional disruption of theRyk gene, e.g., a corticospinal tract (CST)-specific disruption of theRyk gene. In various embodiments, the disrupted Ryk gene includes arecombinant Ryk allele, a selectable marker, frt sites flanking theselectable marker, and loxP sites flanking a portion of the allele. Themarker may be PGK Neo and the loxP sites may flank exons 3-6 of theallele. Thus, the invention also provides an isolated cell derived fromthe transgenic non-human mammal. Also provided is a vector for makingthe transgenic non-human mammal whose genome comprises a heterozygous orhomozygous deletion, inactivation or knock-out of the Ryk gene. Anexemplary vector can include a portion of a Ryk gene, wherein exons 3-6of the Ryk gene are flanked by 3′ and 5′ loxP sites, a selectable markerbetween exon 6 and the 5′ loxP site, and frt sites flanking theselectable marker.

In another aspect, the invention provides a method of producing aknockout mouse with a conditional disruption of the Ryk gene, e.g., aCST-targeted disruption in a Ryk gene. The method includes transfectingthe vector described above into a population of murine embryonic stem(ES) cells, selecting a transfected ES cell which expresses saidselectable marker, introducing said transfected ES cell into an embryoof an ancestor of said mouse, allowing said embryo to develop to term toproduce a chimeric mouse with a conditional knock-out construct in itsgerm line, breeding said chimeric mouse to produce a heterozygous mousewith a conditionally disruptable Ryk gene, and breeding saidheterozygous mouse with a mouse containing a loxP-flanked stop cassettepreventing tdTomato expression only in corticospinal axons to produce amouse with a conditional disruption of the Ryk gene, e.g., aCST-specific disruption in the Ryk gene.

In another aspect, the invention provides a method of screening for anagent that modulates the amount, level and/or activity of atypicalprotein kinases C (aPKC) or MARK2. For example, provided herein is amethod of screening of screening for a therapeutic agent for treating aneurological disease or disorder. The method includes administering atest agent to the transgenic non-human mammal described herein andevaluating the effect of the test agent on at least one of: the amountof atypical protein kinases C (aPKC) or MARK2 protein, the level of aPKCor MARK2 activity or the level of aPKC or MARK2 in at least onedisease-relevant tissue of the transgenic non-human mammal, wherein atleast one of: a decrease in the amount of aPKC protein, an increase inthe amount of MARK2 protein, a decrease in the level of aPKC activity,an increase in the level of MARK2 activity, a reduction in the level ofaPKC, or an increase in the level of MARK2 in at least onedisease-relevant tissue relative to a similar transgenic non-humanmammal that does not receive the test agent indicates the test agent istherapeutic for the neurological disease or disorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are pictorial and graphical diagrams showing that inhibitionof aPKC induces neurite degeneration and neuronal apoptosis. FIG. 1Ashows that endogenous aPKC is localized in cortical neuron cell bodies(E16.5) (arrows) and in SMI-312+ axons (arrowheads). FIGS. 1B-1F showoverexpression of PKCζ-WT, PKCζ-KD or PKCζ-T410A in cerebral corticalneurons (pCIG2-EGFP was used as control). FIG. 1B shows that recombinantPKCζ-WT protein is localized in both neuronal cell bodies (arrows) andprocesses (arrowheads) while PKCζ-KD and PKCζ-T410A proteins are absentin neurites. FIG. 1C shows a higher magnification image showing intactneurites (plain arrows) in a PKCζ-WT transfected neuron and fragmentedneurites in a PKCζ-KD transfected cell (dotted arrows) labeled for EGFP.FIG. 1D shows aCasp3 immunostaining in neurons transfected with theindicated plasmid. FIG. 1E shows the number of transfected neurons withdegenerative neurites (dotted arrows in B) counted over the total numberof EGFP+ neurons and expressed as the % of the control. FIG. 1F showsthe number of apoptotic transfected neurons (aCasp3+ EGFP+) counted overthe total number of EGFP+ neurons. Data represent mean±SEM (n=5experiments in FIG. 1E, n=3 experiments in FIG. 1F. * p<0.05, ** p<0.01,ANOVA with Bonferroni post-test. Scale bars: 200 μm (FIG. 1B), 100 μm(FIG. 1D), 50 μm (FIGS. 1A and 1C).

FIGS. 2A-2H are pictorial and graphical diagrams showing that aPKCinhibition induces rapid axon degeneration that precedes neuronal cellbody death. FIGS. 2A and 2B show axon degeneration in E16.5 cerebralcortical neurons treated with 10 μM myristoylated aPKC PS for 2 h. Theproportion of neurons with degenerative axons was counted over the totalnumber of neurons (SMI-312+ cells,). Plain arrows indicate neurons withintact axons, dotted arrows neurons with beading axons and arrowheadsneurons without axons. FIGS. 2C and 2D show aPKC PS induced axonfragmentation in a dose-(FIG. 2C) and time- (FIG. 2D) dependent manner.FIG. 2E shows aPKC PS treatment reduced phosphorylation of PKCζ-T410.Cell cultures were fixed and immunolabeled with anti-phospho-PKCζ-T410antibody. P-PKCζ-T410 immunoreactivity was measured in neuronal cellbodies (arrows) in neuronal cell cultures incubated with water (Control)or 10 μM aPKC PS for 2 h. FIGS. 2F-2I show axon degeneration induced byaPKC inhibition precedes cell body death. Cortical neurons incubatedwith water (Control) or 10 μM aPKC PS for 2 h, 4 h or 24 h, fixed anddouble-labeled for SMI-312 and TUNEL (FIG. 2F) or activated caspase-3.Plain arrows indicate dead neuronal cell bodies labeled for TUNEL incontrol neurons and neurons treated with aPKC PS. Dotted arrows indicateneurons with beading axons that are not labeled for TUNEL. Graphsrepresent the proportion of neurons with degenerative axons (FIG. 2G)and of dead neurons (TUNEL+(FIG. 2H) or aCasp3+(FIG. 2I)) counted after2 h, 4 h or 24 h of aPKC PS treatment in SMI-312+ neurons. Datarepresent mean±SEM (n=3 experiments). * p<0.05, ** p<0.01, ***p<0.001.FIGS. 2B-2D: ANOVA with Bonferroni post-test; FIGS. 2E and 2G-2I:unpaired Student's t test. Scale bars: 50 μm (FIGS. 2A and 2F), 25 μm(FIG. 2E).

FIGS. 3A-3I are pictorial and graphical diagrams showing that inhibitionof aPKC destabilizes microtubules through regulation of MARK2 activityand Tau phosphorylation. FIGS. 3A and 3B show axon degeneration inducedby aPKC is partially prevented by stabilizing microtubules with taxol.Cortical neurons were incubated with 10 μM taxol for 2 hours prior toand during 2 h treatment of 7 μM aPKC PS. Cells were fixed andimmunolabeled with SMI-312 antibody. The proportion of neurons withintact axons was counted in SMI-312+ neurons (FIG. 3B). Plain arrowsindicate neurons with intact axons. Dotted arrows indicate neurons withbeading axon. Arrowheads indicate neurons without axons. FIGS. 3C-3Fshow the results from Western blottings performed with proteins lysatesfrom E16.5 cerebral cortical cell cultures incubated with water(Control) or 10 μM aPKC PS for 2 hours. Phospho-protein levels wereassessed by densitometric analysis and normalized with the correspondingtotal protein and actin or GAPDH (FIGS. 3C-3E). The level of Glu-tubulinprotein was normalized with GAPDH (FIG. 3F). Lanes 1-3 and lanes 4-6represent triplicates for control and for aPKC PS respectively. FIGS.3G-3I show that Tau-S262A overexpression protects neurons from neuritedegeneration induced by PKCζ-KD overexpression. E16.5 cortical cellscultured for 3 days transfected with the indicated plasmids were fixed 2days after transfection and immunolabeled for EGFP and aCasp3 andcounterstained with DAPI. FIG. 3H shows the number of transfectedneurons with degenerative neurites counted over the total number ofEGFP+ neurons. FIG. 3I shows the number of apoptotic transfected neurons(aCasp3+ EGFP+) counted over the total number of EGFP+ neurons. Datarepresent mean±SEM. FIGS. 3B-3F: n=3 experiments; FIGS. 3H and 3I: n=4experiments. * p<0.05, ** p<0.01, *** p<0.001, FIGS. 3B-3F: UnpairedStudent's t test; H,I: ANOVA with Bonferroni post-test. Scale bars: 50μm (FIG. 3A), 100 μm (FIG. 3G).

FIGS. 4A-4E are pictorial and graphical diagrams showing that aPKCinhibition activates the JNK-cJun signaling pathway. FIGS. 4A-4C showthat JNK phophorylation is increased upon aPKC inhibition. Westernblotting were performed with proteins lysates from E16.5 corticalneurons cultured for 3 days incubated with water (Control) or 10 μM aPKCPS for 1 h or 2 h. FIGS. 4B and 4C show phospho-protein levels assessedby densitometric analysis. Levels of phospho-proteins were normalizedwith the corresponding total protein and GAPDH. FIGS. 4D and 4E showthat cortical neuronal cultures incubated with water (Control) or 10 μMaPKC PS for 1 h were immunolabled for P-JNK T183/T185 (D) or P-c-Jun S63(FIG. 4E). Arrows indicate increased immunoreactivity in neurons treatedwith aPKC PS compared to control neurons. Data represent mean±SEM. n=3experiments/group, * p<0.05, ** p<0.01, unpaired Student's t test. Scalebar: 100 μm.

FIGS. 5A-5F are pictorial and graphical diagrams showing that Rykpromotes axonal degeneration induced by aPKC inhibition. FIGS. 5A and 5Bshow that aPKC activity is increased in Ryk KO mouse cortical neuronalcell cultures. Western blotting were performed with proteins lysatesfrom cortical neurons from E16.5 Ryk wild type, heterozygous and KOembryos cultured for 3 days in vitro. Levels of P-PKCζ-T410 wereassessed by densitometric analysis and normalized with total PKCζ andGAPDH. FIGS. 5C and 5D show that monoclonal Ryk antibody blocksdegeneration. E16.5 cortical neurons were incubated with monoclonalmouse anti-Ryk antibody (50 μg/ml or 100 μg/ml) or normal mouse IgG (100μg/ml) for 2 hours prior to and during 2 h treatment of 7 μM aPKC PS.Cells were fixed and immunolabeled for SMI-312. FIG. 5D shows theproportion of SMI-312+ neurons with degenerative axons. FIGS. 5E and 5Fshow reduced degeneration with Ryk knockout mouse. Cortical neuronsindividually dissociated from E16.5 WT (Ryk^(+/+)) and Ryk KO embryos(Ryk^(−/−)) were incubated with 7 μM aPKC PS for 2 h. Cells were fixedand immunolabeled for SMI-312. FIG. 5F shows the proportion of SMI-312+neurons with degenerative axons in Ryk^(+/+) and Ryk^(−/−) neuronstreated with aPKC PS or water (control). Plain arrows indicate neuronswith intact axons, dotted arrows neurons with beading axons andarrowheads neurons without axons. Data represent mean±SEM (FIG. 5A:Ryk^(+/+): n=3 embryos, Ryk^(+/−) n=3 embryos, Ryk^(−/−): n=4 embryos;D: n=5 experiments; FIG. 5F: Ryk^(+/+): n=4 embryos, Ryk^(−/−): n=6embryos). * p<0.05, ** p<0.001, *** p<0.001; FIGS. 5B and 5F: UnpairedStudent's t test, D: ANOVA with Bonferroni post-test. Scale bars: 100μm.

FIGS. 6A-6E are pictorial and graphical diagrams showing geneticinteraction between Ryk and Frizzled3 in neuronal cell death. FIG. 6Ashows a lower magnification of a WT brain section (Ryk^(+/+)) showingthe localization of the retrosplenial (RSP) cortex. FIGS. 6B and 6C showthat aCasp3⁺ cell number is decreased in Ryk^(−/−) embryos compared toRyk^(+/+) mice. FIGS. 6D and 6E show that aCasp3⁺ cell number isincreased in the RSP of Frizzled3^(−/−) embryos compared toFrizzled3^(+/+) and Frizzled3^(+/−) mice. Ryk knock-down attenuates celldeath in the RSP of Frizzled3KO mice. Data represent mean±SEM(Ryk^(+/+): n=6 embryos, Ryk^(+/−): n=4 embryos, Ryk^(−/−): n=5 embryos,Frizzled3^(+/+): n=4 embryos, Frizzled3^(+/−): n=6 embryos,Frizzled3^(−/−): n=8 embryos, Frizzled3^(−/−) Ryk^(+/−): n=5 embryos). *p<0.05, ** p<0.01, ** p<0.001. FIG. 6C: Student's t test, E: ANOVA withBonferroni post-test. HIPP: hippocampus, LV: lateral ventricle, RSP:retrosplenial cortex. Scale bars: 500 μm (FIG. 6A), 200 μm (FIGS. 6B and6D).

FIGS. 7A and 7B are pictorial diagrams showing a model of aPKC action onaxonal integrity and neuronal cell survival. FIG. 7A shows that innormal conditions when aPKC is expressed and active, aPKC inhibits MARK2activity through phosphorylation on T585. Phosphorylation state of Tauis low and Tau binds to and stabilizes microtubules, maintaining axonintegrity and neuronal cell survival. FIG. 7B shows that when aPKCkinase activity is reduced or inhibited, MARK2 activity increases andphosphorylates Tau in its microtubule binding domain on S262.Hyperphosphorylated Tau detach from microtubules which destabilizesmicrotubules. Disruption of microtubules activates the stress kinaseJNK/SAPK pathway, leading to axonal degeneration and neuronal celldeath.

FIGS. 8A-8F are pictorial and graphical diagrams showing that Rykconditional deletion enhances motor function recovery from spinal cordinjury. FIG. 8A shows a timeline outlining experimental details ofbilateral cervical level S (CS) dorsal column lesion. FIGS. 8B-8C showgeneration of Ryk conditional allele. FIG. 8B shows that exons 3-6 wereflanked with loxP sites. FIG. 8C shows the results of a Western blot ofpostnatal day 7 motor cortex extract from mice infected at postnatal day0 with AAV2/1 synapsin Cre. Full-length blot presented in FIGS. 23A and23B. FIGS. 8D and 8E show a schematic showing the level of the CS lesionin relation to motor neuron pools for distinct forelimb muscle groups(adapted from McKenna, Prusky, and Whishaw, 2000). FIG. 8F showsbehavioral performance on forelimb reach skilled food-pellet retrievaltask shows enhanced recovery after Ryk conditional deletion in bilateralmotor cortex (n=2S mice (control), 17 mice (Ryk cKO), from 21 litters,repeated measures ANOVA P=0.0003, F(1,40)=16.0102). Data presented asmean±s.e.m.

FIGS. 9A-9H are pictorial and graphical diagrams showing Ryk conditionaldeletion enhances corticospinal axon sprouting after spinal cord injury.FIGS. 9A and 9B are representative images of tdTomato-labeled CST axonsfrom eight serial sagittal cryosections spaced 140 μm apart superimposedover GFAP astroglial staining at the center of injury (1 experiment,n=12 mice/group, from 11 litters; compass showing dorsal (D), ventral(V), rostral (R), and caudal (C)). Mice with Ryk conditional deletionhad greater levels of collateralization both rostral and caudal to thelesion than control mice (one-tailed t-test *P<O.OS). FIGS. 9C and 9Dshow higher magnification of single confocal planes from boxed regions(1.5 mm caudal to the lesion site) indicated in FIGS. 9A and 9B,respectively. FIG. 9E shows the sum of tdTomato labeled axons(normalized to pyramidal labeling) over 3 mm rostral to lesion relativeto control in the dorsal columns (n=12 mice/group, one-tailed t-testP=0.12, t(21)=1.198) and in the gray matter. Mice with Ryk conditionaldeletion had greater levels of collateralization both rostral and caudalto the lesion than control mice (n=12 mice/group, one-tailed t-test*P<O.OS: rostral P=0.0499 t(19)=1.730, caudal P=0.0397 t(19)=1.855).FIGS. 9F-9H show the distribution of corticospinal axons (axon index isthresholded pixels at every 0.411 μm in 8 total saggital spinal cordcryosections divided by thresholded pixels in transverse pyramids)within the dorsal columns (FIG. 9F) or spinal gray matter (FIGS. 9G and9H). FIG. 9H is a magnified view of rostral collaterals from FIG. 9G. CSinjury site is at 0 μm, rostral is represented with negative numbers,caudal with positive. Data in FIG. 9E is presented as median withinter-quartile range, data in FIGS. 9F-9H is presented as mean±s.e.m.

FIGS. 10A-10D are pictorial and graphical diagrams showing changes ofcorticospinal connectivity after CS dorsal column lesion. FIGS. 10A and10B show media-lateral distribution of corticospinal axons shows highestincrease in collaterals proximal to the main, dorsal corticospinal tract(regions I and II) after Ryk conditional deletion (n=12 mice/group,one-tailed t-test *P<O.OS: II rostral P=0.0225 t(19)=2.146, II caudalP=0.0295 t(21)=1.996, I caudal P=0.0059 417)=2.819). FIG. 10C shows bothcontrol and Ryk conditional deletion mice showed pre-synaptic densities(vGlut1 colocalization with tdTomato-labeled corticospinal axons) at 600μm rostral to the CS injury site (1 experiment, n=9 mice/group). FIG.10D shows media-lateral distribution of corticospinal innervation at 600μm rostral to CS injury site. All data presented as median andinter-quartile range.

FIGS. 11A-11F are pictorial and graphical diagrams showing thatsecondary injury at cervical level 3 eliminates enhanced recovery. FIG.11A is a timeline outlining experimental details of secondary C3 lesionexperiments following recovery from bilateral CS dorsal column lesion.FIG. 11B is a schematic of secondary C3 injury, above the level ofincreased pre-synaptic density shown in (FIGS. 10C-10E). FIG. 11C showsbehavioral performance on forelimb reach skilled food-pellet retrievaltask shows elimination of enhanced recovery after Ryk conditionaldeletion by second C3 dorsal column lesion (1 experiment, n=8 (controlsham), 7 (control C3), 6 (Ryk cKO sham & C3) mice, mice from 14 litters,ANOVA P=0.0102 F(3)=4.7432, Bonferroni corrected t-test *P<0.05:1. RykcKO sham v. control sham P=0.0106, 2. Ryk cKO sham v. Ryk cKO C3P=0.0092). FIGS. 11D-11F show secondary C3 dorsal column lesioneliminated enhanced levels of collateralization in Ryk conditionaldeleted mice. FIG. 11D shows representative images of tdTomato-labeledCST axons from 8 serial sagittal cryosections spaced 140 μm apartsuperimposed over GFAP astroglial staining at the center of injury (1experiment, 7 control, 6 Ryk cKO mice). FIGS. 11E and 11F showdistribution of corticospinal axons (axon index as described above)within the dorsal columns (FIG. 11E) or spinal gray matter (FIG. 11F).C3 injury site is at 0 μm, rostral is represented with negative numbers,caudal with positive. Data in FIG. 11C presented as median andinter-quartile range, data in FIGS. 11E and 11F are presented asmean±s.e.m.

FIGS. 12A-12I are pictorial and graphical diagrams showing thatmonoclonal Ryk antibody infusion promotes functional recovery fromspinal cord injury. FIG. 12A is a schematic showing antibody infusion byintrathecal catheterization. FIG. 12B shows behavioral performance onforelimb reach skilled food-pellet retrieval task shows enhancedrecovery in rats infused with Ryk monoclonal antibody for 28 daysstarting at time of injury (n=6 rats (IgG control), 5 rats (Rykmonoclonal), repeated measures ANOVA P=0.0354, F(1,9)=6.113). FIG. 12Cshows behavioral performance on skilled locomotor grid crossing task wasnot affected by Ryk monoclonal antibody infusion. FIG. 12D shows thatRyk monoclonal recognizes full-length Ryk protein expressed intransfected COS-7 cells by Western and immunocytochemistry. FIGS.12E-12H show that BDA-labeled corticospinal axons in rats infused withRyk monoclonal antibody had greater levels of collateralization thancontrol mouse IgG infused rats. FIGS. 12E and 12F are images ofBDA-labeled CST axons from 6 serial sagittal cryosections spaced 280 μmapart superimposed over GFAP astroglial and NG2 staining at the centerof injury. FIGS. 12G and 5H show a distribution of corticospinal axons(axon index is thresholded pixels at every 0.741 μm in 6 total saggitalspinal cord cryosections divided by thresholded pixels in transversepyramids) within the dorsal columns (FIG. 12G) or spinal gray matter(FIG. 12H). CS injury site is at 0 μm, rostral is represented withnegative numbers, caudal with positive. FIG. 12I shows the sum ofnormalized axon collaterals over 5 mm relative to control. Rats infusedwith Ryk monoclonal antibody had greater levels of collateralizationboth rostral and caudal to the lesion than control mouse IgG infusedrats (n=6 rats (IgG control), 5 rats (Ryk monoclonal), one-tailed t-test585 *P<0.05: rostral P=0.0446 46)=2.000, caudal P=0.0196 46)=2.594).Data in FIGS. 12B, 12C, 12G, and 12H is presented as mean±s.e.m., datain FIG. 12I is presented as median and inter-quartile range.

FIGS. 13A-13C are graphical diagrams showing cortical mapre-organization during recovery from spinal cord injury. FIG. 13A is atimeline outlining experimental details of optogenetic mapping withweekly behavioral testing following bilateral CS dorsal column lesion.FIG. 13B is a topographic representation of elbow flexor and extensoractivation, relative to bregma (*) prior to and 3 days, 4 weeks, and 8weeks after CS dorsal column lesion. Data presented as total number ofmice responding with evoked movements at each location, lighter colorindicates a larger number of mice are responsive at a given location.Each tic mark represents 300 μm. FIG. 13C shows that subsequent C3dorsal column lesion disrupts remodeled circuitry, while subsequentpyradmidotomy eliminates unilateral evoked motor output. Both measuredat 3 days after injury.

FIGS. 14A-14F are graphical and pictorial diagrams showing forelimbmotor map representations infiltrate quiescent former hindlimb corticalareas. FIG. 14A is a cortical map re-organization from spinal cordinjury in mice that received weekly training demonstrates how mapcentroids shift after injury. Size of the marker is proportional to thepercentage of mice with evoked motor movements of a given muscle group.FIGS. 14B and 14C show elbow extensor motor maps shift caudal and medialtowards cortex originally occupied by hindlimb representations. FIGS.14D and 14E show that mice with Ryk conditional deletion have a greaterproportion devoted to elbow flexor activation at 4 weeks post-CS lesion(FIG. 14D) and conversely a smaller proportion of the motor cortexdevoted to extensor activation (FIG. 14E) (n=10 (control) 11 (Ryk cKO)mice, one-tailed t-test *P<0.05: elbow flexion P=0.0347 t(19)=1.925,elbow extension P=0.0460 t(16)=−1.791, data presented as mean±s.e.m.).FIG. 14F is a model for recruitment of ectopic cortical motor regionsmediated by axon plasticity. After dorsal column injury, the immediateexpansion of forelimb regions above the level of injury is likelymediated by lateral connectivity within the motor cortex. Increasedaxonal plasticity and connectivity after Ryk conditional deletion likelydrives the formation of novel, ectopic areas of forelimb motor cortex.

FIGS. 15A-15D are graphical diagrams showing that cortical mapre-organization and functional recovery from spinal cord injury aredependent upon rehabilitative training. FIG. 15A is a timeline outliningexperimental details of optogenetic mapping with only terminalbehavioral testing at 8 weeks post-injury. FIG. 15B is a topographicrepresentation of elbow flexor and extensor activation, relative tobregma (*) prior to and 3 days, 4 weeks, and 8 weeks after CS dorsalcolumn lesion in the absence of weekly behavioral testing. FIG. 15Cshows that at 8 weeks after CS dorsal column lesion, mice with weeklybehavioral testing, both Ryk cKO and controls, performed better thanthose only tested at 8 weeks (n=10 (control weekly testing), 11 (Ryk cKOweekly testing), 5 (control & Ryk cKO 8 wk only testing) mice, ANOVAP=0.0037 F(3)=5.7157, Bonferroni corrected t-test *P<0.05:1. Ryk cKOweekly v. 8 week only testing P=0.0277, 2. control weekly v. 8 week onlytesting P=0.0346, data presented as median and inter-quartile range).FIG. 15D shows that in animals with weekly behavioral testing (blackXs), there was a strong correlation of wrist movement and skilledforelimb reach performance, regardless of injury or genotype (n=84measurements (4 time points, 21 mice), bivariate Pearson correlation P,P<0.0001 p=0.665). Light blue is density ellipse (a=0.95). Mice with noweekly behavioral testing are shown in red.

FIG. 16 is a pictorial diagram showing a series of sample frames from aforelimb read video. This example at 13 weeks after C5 dorsal columnlesion (1 week after C3 sham operation) demonstrates the recovery ofskilled forelimb grasp. Successful reach requires the use of a graspingmotion as a sweeping motion would result in the pellet being dropped ineither the gap between the food pellet platform (black) and the mainenclosure, or through the wire-frame floor of the enclosure.

FIGS. 17A and 17B are pictorial diagrams showing Ryk monoclonal antibodyinfusion in rats. FIG. 17A shows the specificity of Ryk monoclonalantibody. Full-length blot presented in FIGS. 23A and 23B. FIG. 17B is atimeline outlining experimental details of Ryk monoclonal antibodyinfusion after bilateral C5 dorsal column lesion in rats.

FIG. 18 is a graphical diagram showing that axon collateralizationincreased after Ryk monoclonal antibody infusion caudal to the injury.Rats infused for 28 days with Ryk monoclonal antibody had greater levelsof collateralization caudal to the lesion than control IgG infused rats(n=6 (IgG control) 5 (Ryk monoclonal) rats, one-tailed t-test *P=0.0196P=0.0196 t(6)=2.594, data presented as mean±s.e.m.). Injury site is at 0μm, caudal is represented with positive numbers. Axon index isthresholded pixels in sagittal spinal cord divided by thresholded pixelsin transverse pyramids.

FIG. 19 is a pictorial diagram showing an optogenetic mapping example.Sedated mice with unilateral cranial windows were stimulated with 470 nmLED by fiber optic cable to evoke muscle movements. Two examples ofmotor maps from one animal, pre- and 3 days post-CS dorsal column lesionare shown.

FIG. 20 is a graphical diagram showing the proportion of motor cortexoccupied by characterized motor output changes over time in response toweekly training and Ryk conditional deletion

FIG. 21 is a graphical diagram showing recovery of skilled forelimbreach for mice used in cortical mapping experiments. Behavioralperformance on skilled forelimb reach task shows enhanced recovery afterRyk conditional deletion in contralateral motor cortex (weeks 1-8 afterC5 lesion, n=10 (control) 11 (Ryk cKO) mice, repeated measures ANOVAP=0.0304 F(1,19)=5.472). Secondary C3 eliminates enhanced recovery afterRyk conditional deletion (n=9 (control) 8 (Ryk cKO)), while unilateralpyramidotomy (n=8 (control) 7 (Ryk cKO)) completely ablates the abilityof mice to perform the task. Data presented as mean±s.e.m.

FIG. 22 is a graphical diagram showing that proportions of motor cortexoccupied by characterized motor output are relatively stable in theabsence of training after injury.

FIGS. 23A and 23B are pictorial diagrams showing the results of fullWestern blots from FIG. 8C and FIG. 17A. FIG. 23A shows the specificityof Ryk monoclonal antibody from FIG. 17A. FIG. 23B shows a Western blotof postnatal day 7 motor cortex extract from mice infected at postnatalday 0 with AAV2/1 synapsin Cre from FIG. 8C. E18.5 cortex from twoseparate Ryk KO embryonic mouse cortices as control in right two lanes.GAPDH loading control from same blot.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that an anti-Ryk antibodyor antibody fragment that specifically binds to a binding domain of Wntinhibits Wnt-Ryk signaling. As such, the present invention providesmethods for modulating neuron degeneration and neuron guidance using theanti-Ryk antibody or antibody fragment. Thus, the anti-Ryk antibody orantibody fragment can be used to treat a neurological disease ordisorder, e.g., a neurodegenerative disease or disorder, in a subjecthaving or being at risk of developing the neurological disease ordisorder, e.g., a neurodegenerative disease or disorder, and/or to treatspinal cord injury (SCI) in a subject.

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

The term “comprising,” which is used interchangeably with “including,”“containing,” or “characterized by,” is inclusive or open-ended languageand does not exclude additional, unrecited elements or method steps. Thephrase “consisting of” excludes any element, step, or ingredient notspecified in the claim. The phrase “consisting essentially of” limitsthe scope of a claim to the specified materials or steps and those thatdo not materially affect the basic and novel characteristics of theclaimed invention. The present disclosure contemplates embodiments ofthe invention compositions and methods corresponding to the scope ofeach of these phrases. Thus, a composition or method comprising recitedelements or steps contemplates particular embodiments in which thecomposition or method consists essentially of or consists of thoseelements or steps.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably herein. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made. Treatment includespreventing the disease, that is, causing the clinical symptoms of thedisease not to develop by administration of a protective compositionprior to the induction of the disease; suppressing the disease, that is,causing the clinical symptoms of the disease not to develop byadministration of a protective composition after the inductive event butprior to the clinical appearance or reappearance of the disease;inhibiting the disease, that is, arresting the development of clinicalsymptoms by administration of a protective composition after theirinitial appearance; preventing re-occurring of the disease and/orrelieving the disease, that is, causing the regression of clinicalsymptoms by administration of a protective composition after theirinitial appearance.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an active agent sufficient to induce a desiredbiological result. That result may be alleviation of the signs,symptoms, or causes of a disease, or any other desired alteration of abiological system. The term “therapeutically effective amount” is usedherein to denote any amount of the formulation which causes asubstantial improvement in a disease condition when applied to theaffected areas repeatedly over a period of time. The amount will varywith the condition being treated, the stage of advancement of thecondition, and the type and concentration of formulation applied.Appropriate amounts in any given instance will be readily apparent tothose skilled in the art or capable of determination by routineexperimentation.

The term “pharmaceutically acceptable salt” refers to salts derived froma variety of organic and inorganic counter ions well known in the artand include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate,maleate, oxalate and the like.

A “subject,” “individual,” or “patient,” is used interchangeably herein,which refers to a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murines, simians,humans, farm animals, sport animals, and pets. Tissues, cells and theirprogeny of a biological entity obtained in vitro or cultured in vitroare also encompassed.

As used herein, “promote” or “increase,” or “promoting” or “increasing”are used interchangeably herein. These terms refer to the increase in ameasured parameter (e.g., activity, expression, signal transduction,neuron degeneration) in a treated cell (tissue or subject) in comparisonto an untreated cell (tissue or subject). A comparison can also be madeof the same cell or tissue or subject between before and aftertreatment. The increase is sufficient to be detectable. In someembodiments, the increase in the treated cell is at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-foldor more in comparison to an untreated cell.

As used herein, “inhibit,” “prevent” or “reduce,” or “inhibiting,”“preventing” or “reducing” are used interchangeably herein. These termsrefer to the decrease in a measured parameter (e.g., activity,expression, signal transduction, neuron degeneration) in a treated cell(tissue or subject) in comparison to an untreated cell (tissue orsubject). A comparison can also be made of the same cell or tissue orsubject between before and after treatment. The decrease is sufficientto be detectable. In some embodiments, the decrease in the treated cellis at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, orcompletely inhibited in comparison to an untreated cell. In someembodiments the measured parameter is undetectable (i.e., completelyinhibited) in the treated cell in comparison to the untreated cell.

The term “selective inhibition” or “selectively inhibit” as referred toa biologically active agent refers to the agent's ability topreferentially reduce the target signaling activity as compared tooff-target signaling activity, via direct or indirect interaction withthe target.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Naturally encoded amino acids arethe 20 common amino acids (alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

A conservative substitution may include substitution such as basic forbasic, acidic for acidic, polar for polar, etc. The sets of amino acidsthus derived are likely to be conserved for structural reasons. Thesesets can be described in the form of a Venn diagram (Livingstone C. D.and Barton G. J. (1993) “Protein sequence alignments: a strategy for thehierarchical analysis of residue conservation” Comput. Appl Biosci. 9:745-756; Taylor W. R. (1986) “The classification of amino acidconservation” J. Theor. Biol. 119; 205-218). Conservative substitutionsmay be made, for example, according to the table below which describes agenerally accepted Venn diagram grouping of amino acids.

TABLE 1 Grouping of amino acids Characteristic Set CharacteristicSub-set Hydrophobic F W Y H K M I L V A G C Aromatic AliphaticF W Y H I L V Polar W Y H K R E D C S T N Charged PositiveH K R E D H K R Q Charged Negative E D Small V C A G S P T N D TinyA G S

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (e.g., a polypeptide of the invention), which doesnot comprise additions or deletions, for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same sequences. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity overa specified region, or, when not specified, over the entire sequence),when compared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. The invention provides polypeptides that are substantiallyidentical to the polypeptides, respectively, exemplified herein, as wellas uses thereof including, but not limited to, use for treating orpreventing neurological diseases or disorders, e.g., neurodegenerativediseases or disorders, and/or treating SCI. Optionally, the identityexists over a region that is at least about 50 nucleotides in length, ormore preferably over a region that is 100 to 500 or 1000 or morenucleotides in length, or the entire length of the reference sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form, andcomplements thereof. The term encompasses nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, and non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

As used herein, the term “dominant negative mutant” of a protein refersto a mutant polypeptide or nucleic acid, which lacks wild-type activityand which, once expressed in a cell wherein a wild-type of the sameprotein is also expressed, dominates the wild-type protein andeffectively competes with wild type proteins for substrates, ligands,etc., and thereby inhibits the activity of the wild type molecule. Thedominant negative mutant can be a polypeptide having an amino acidsequence substantially similar (i.e., at least about 75%, about 80%,about 85%, about 90%, about 95% similar) to the wild type protein. Thedominant negative mutant can also be a polypeptide comprising a fragmentof the wild type protein, e.g., the C-domain of the wild-type protein.The dominant negative mutant can be a truncated form of the wild typeprotein.

Mouse Model

As used herein, “transgenic organism” refers to an animal in whichexogenous DNA has been introduced while the animal is still in itsembryonic stage. In most cases, the transgenic approach aims at specificmodifications of the genome, e.g., by introducing whole transcriptionalunits into the genome, or by up- or down-regulating or mutatingpre-existing cellular genes. The targeted character of certain of theseprocedures sets transgenic technologies apart from experimental methodsin which random mutations are conferred to the germline, such asadministration of chemical mutagens or treatment with ionizing solution.A transgenic organism can include an organism which has a gene knockoutor may result for inducing a genetic mutation.

A “genetic knock out” refers to partial or complete suppression of theexpression of a protein encoded by an endogenous DNA sequence in a cell.The “knockout” can be affected by targeted deletion of the whole or partof a gene encoding a protein. Alternatively, the transgenic organism canbe obtained by the targeted mutation of a functional protein in anembryonic stem cell. As a result, the deletion or mutation may preventor reduce the expression of the protein in any cell in the whole animalin which it is normally expressed, or results in the expression of amutant protein having a biological function different than thenormal/wild-type protein.

The term “knockout animal” and “transgenic animal”, refer to atransgenic animal wherein a given gene has been suppressed or mutated byrecombination with a targeting vector. It is to be emphasized that theterm is intended to include all progeny generations. Thus, the founderanimal and all F1, F2, F3, and so on, progeny thereof are included.

As used herein, the phrase “conditional knockout,” or “cKO,” when usedto describe a non-human transgenic mammal such as a mouse, refers tomice containing a knock-out of a specific gene in a certain tissue. Thecreation of a genetically engineered cKO mouse involves insertingspecific DNA sequences, such as a knock-out construct/vector, into themouse DNA. The inserted sequences are recognized by two DNA specificenzymes, frt recombinase (also known as flippase) and Cre recombinase,not normally present in mice. Cre recombinase recognition sites aretermed loxP sites and flippase recognition sites are termed frt sites.Each of these enzymes can cut and remove a DNA sequence that is flankedby its recognitions sites. This can lead to disruption of gene functionif a functional DNA sequence of the gene of interest is removed. Inaddition, a selectable marker gene is inserted into the mouse, theintroduction of which allows selection of embryonic mouse cells (stemcells) that contain the Cre recombination or flippase recognition sites.The resultant mouse is a conditional knockout mouse.

A knock-out construct is a nucleic acid sequence, such as a DNAconstruct, which, when introduced into a cell, results in suppression(partial or complete) of expression of a polypeptide or protein encodedby endogenous DNA in the cell. An exemplary knock-out construct isprovided herein. This construct contains a loxP site 5′ to exon 3 and 3′to exon 6 of the Ryk gene, a selectable marker cassette and a loxP site3′ to the selectable marker cassette. The selectable marker cassettecomprises frt sites 5′ and 3′ to the selectable marker and is betweenthe 3′ frt site and the selectable marker gene. Suitable selectablemarkers include, but are not limited to, neomycin, puromycin andhygromycin.

Animals containing more than one transgenic construct and/or more thanone transgene expression construct may be prepared in any of severalways. An exemplary manner of preparation is to generate a series ofanimals, each containing one of the desired transgenic phenotypes. Suchanimals are bred together through a series of crosses, backcrosses andselections, to ultimately generate a single animal containing alldesired transgenic traits and/or expression constructs, where the animalis otherwise congenic (genetically identical) to the wild type exceptfor the presence of the construct(s) and/or transgene(s).

Embryonic stem (ES) cells are typically selected for their ability tointegrate into and become part of the germ line of a developing embryoso as to create germ line transmission of the transgene. Thus, any EScell line that can do so is suitable for use herein. ES cells aregenerated and maintained using methods well known to the skilledartisan, such as those described by Doetschman et al. (1985) J. Embryol.Exp. Mol. Biol. 87:27-45). Any line of ES cells can be used, however,the line chosen is typically selected for the ability of the cells tointegrate into and become part of the germ line of a developing embryoso as to create germ line transmission of the transgenic/knockoutconstruct. Thus, any ES cell line that is believed to have thiscapability is suitable for use herein. One mouse strain that istypically used for production of ES cells, is the 129J strain. AnotherES cell line is murine cell line D3 (American Type Culture Collection,catalog no. CKL 1934). Still another ES cell line is the WW6 cell line(Ioffe et al. (1995) PNAS 92:7357-7361). The cells are cultured andprepared for knockout construct insertion using methods well known tothe skilled artisan, such as those set forth by Robertson in:Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J.Robertson, ed. IRL Press, Washington, D.C. (1987)); by Bradley et al.(1986) Current Topics in Devel. Biol. 20:357-371); and by Hogan et al.(Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1986)).

Introduction of the knock-out construct into ES cells may beaccomplished using a variety of methods well-known in the art,including, for example, electroporation, microinjection, and calciumphosphate treatment. For introduction of the DNA sequence, the knock-outconstruct DNA is added to the ES cells under appropriate conditions forthe insertion method chosen. If the cells are to be electroporated, theES cells and construct DNA are exposed to an electric pulse using anelectroporation machine (electroporator) and following themanufacturer's guidelines for use. After electroporation, the cells areallowed to recover under suitable incubation conditions. The cells arethen screened for the presence of the knockout construct. Screening forcells which contain the transgene (homologous recombinants) may be doneusing a variety of methods. For example, as described herein, cells canbe processed as needed to render DNA in them available for screeningwith specific probes by polymerase chain reaction (PCR).

Once appropriate ES cells are identified, they are introduced into anembryo using standard methods. They can be introduced usingmicroinjection, for example. Embryos at the proper stage of developmentfor integration of the ES cell to occur are obtained, such as byperfusion of the uterus of pregnant females. For example, mouse embryosat 3-4 days development can be obtained and injected with ES cells usinga micropipet. After introduction of the ES cell into the embryo, theembryo is introduced into the uterus of a pseudopregnant female mouse.The stage of the pseudopregnancy is selected to enhance the chance ofsuccessful implantation. In mice, 2-3 days pseudopregnant females areappropriate.

Successful incorporation of ES cells into implanted embryos results inoffspring termed chimeras. Chimeras capable of germline transmission ofthe mutant allele are identified by standard methods. Chimeras are bredand the resulting progeny are screened for the presence of the desiredalteration (e.g., the modified recombinant Ryk allele). This may bedone, for example, on the basis of coat color or by obtaining DNA fromoffspring (e.g., tail DNA) to assess for the transgene, using knownmethods (e.g., Southern analysis, dot blot analysis, PCR analysis).Transgene expression may also be assessed (e.g., to determine if areplacement construct is expressed) by known methods, such as northernanalysis or PCR analysis. Southern hybridization or PCR analysis ofprogeny DNA (e.g., tail DNA) may be conducted to identify desiredgenotypes. A suitable technique for obtaining completely ES cell derivedtransgenic non-human organisms is described in WO 98/06834, incorporatedherein by reference.

In various embodiments, the cKO mice disclosed herein include at leastthree elements: (1) at least two enzyme-specific recognition sitesflanking a critical portion of the target gene; (2) a gene encoding aselection marker such as, but not limited to neomycin; and (3) at leasttwo enzyme-specific recognition sites flanking a selection marker genefor easy removal upon breeding with specific mouse strains. In anon-limiting example, exons 3-6 of the target gene has been designatedas the critical portion. In one embodiment the enzyme-specificrecognition sites flanking the critical portion of the target gene areloxP sites. In another embodiment, the enzyme-specific recognition sitesflanking the selection marker gene are frt sites.

As mentioned above, the homologous recombination of the above described“knock-out” and/or “knock in” constructs is sometimes rare and such aconstruct can insert non-homologously into a random region of the genomewhere it has no effect on the gene which has been targeted for deletion,and where it can potentially recombine so as to disrupt another genewhich was otherwise not intended to be altered. Such non-homologousrecombination events can be selected against by modifying theabove-mentioned targeting vectors so that they are flanked by negativeselectable markers at either end (particularly through the use of thediphtheria toxin gene, thymidine kinase gene, the polypeptide product ofwhich can be selected against in expressing cell lines in an appropriatetissue culture medium well known in the art—e.g., one containing a drugsuch as ganciclovir. Non-homologous recombination between the resultingtargeting vector comprising the negative selectable marker and thegenome will usually result in the stable integration of one or both ofthese negative selectable marker genes and hence cells which haveundergone non-homologous recombination can be selected against by growthin the appropriate selective media (e.g., media containing a drug suchas ganciclovir). Simultaneous selection for the positive selectablemarker and against the negative selectable marker will result in a vastenrichment for clones in which the construct has recombined homologouslyat the locus of the gene intended to be mutated. The presence of thepredicted chromosomal alteration at the targeted gene locus in theresulting stem cell line can be confirmed by means of Southern blotanalytical techniques which are well known to those familiar in the art.Alternatively, PCR can be used.

Other methods of making transgenic animals are also generally known.See, for example, Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependenttransgenic organisms can also be generated, e.g., by homologousrecombination to insert target sequences, such that tissue specificand/or temporal control of inactivation of a Ryk gene can be controlledby recombinase sequences.

Accordingly, in one aspect, the invention provides a transgenicnon-human mammal such as a mouse whose genome comprises a heterozygousor homozygous deletion, inactivation or knock-out of the Ryk gene andmethods of making the same. In various embodiments, the mouse has thephenotype Frizzled3^(−/−) Ryk^(+/−). In various embodiments, the mousecontains a corticospinal tract (CST)-specific disruption of the Rykgene. In various embodiments, the disrupted Ryk gene includes arecombinant Ryk allele, a selectable marker, frt sites flanking theselectable marker, and loxP sites flanking a portion of the allele. Themarker may be PGK Neo and the loxP sites may flank exons 3-6 of theallele. Also provided is an isolated cell derived from the transgenicnon-human mammal.

Neurons

As used herein, the term “neuron” include a neuron and a portion orportions thereof (e.g., the neuron cell body, an axon, or a dendrite).The term “neuron” as used herein denotes nervous system cells thatinclude a central cell body or soma, and two types of extensions orprojections: dendrites, by which, in general, the majority of neuronalsignals are conveyed to the cell body, and axons, by which, in general,the majority of neuronal signals are conveyed from the cell body toeffector cells, such as target neurons or muscle. Neurons can conveyinformation from tissues and organs into the central nervous system(afferent or sensory neurons) and transmit signals from the centralnervous systems to effector cells (efferent or motor neurons). Otherneurons, designated interneurons, connect neurons within the centralnervous system (the brain and spinal column). Certain specific examplesof neuron types that may be subject to treatment or methods according tothe invention include cerebellar granule neurons, dorsal root ganglionneurons, and cortical neurons.

The term “neuronal degeneration” is used broadly and refers to anypathological changes in neuronal cells, including, without limitation,death or loss of neuronal cells, any changes that precede cell death,and any reduction or loss of an activity or a function of the neuronalcells. The pathological changes may be spontaneous or may be induced byany event and include, for example, pathological changes associated withapoptosis. The neurons may be any neurons, including without limitationsensory, sympathetic, parasympathetic, or enteric, e.g., dorsal rootganglia neurons, motor neurons, and central neurons, e.g., neurons fromthe spinal cord. Neuronal degeneration or cell loss is a characteristicof a variety of neurological diseases or disorders, e.g.,neurodegenerative diseases or disorders. In some embodiments, the neuronis a sensory neuron. In some embodiments, the neuron is a motor neuron.In some embodiments, the neuron is a damaged spinal cord neuron.

In some embodiments, degeneration occurs in a portion of the neuron suchas the neuron cell body, an axon, or a dendrite. Accordingly, thedegeneration can be inhibited in the degenerated portion or portions ofthe neuron. In some embodiments, the degeneration of an axon of theneuron is inhibited. In some embodiments, the degeneration of a cellbody of the neuron is inhibited. The axon can be an axon of any neuron.For example, in some embodiments, the axon is a spinal cord commissuralaxon, or an upper motor neuron axon, or a central nervous system axon.

As described herein, the disclosed methods can be carried out in vivo,such as in the treatment of neurodegenerative diseases, neurologicaldisorders or injuries to the nervous system. The methods can also becarried out in vitro or ex vivo, such as in laboratory studies of neuronfunction and in the treatment of nerve grafts or transplants.Accordingly, in some embodiments, the neuron forms part of a nerve graftor a nerve transplant. In some embodiments, the neuron is ex vivo or invitro. In some embodiments, the nerve graft or the nerve transplantforms part of an organism, human or non-human (e.g., mammal, primate,rat, mouse, rabbit, bovine, dog, cat, pig, etc.).

Axon Degeneration

Axon degeneration is a common feature in many neurological andneurodegenerative diseases/disorders and in traumatic injuries. Studiesindicate that it can occur independent of and before the death ofneuronal cell bodies. However, the molecular and cellular mechanismsunderlying axonal degeneration and protection are still unclear.Elucidating the degeneration pathways that are activated or theprotection pathways that are inactivated during axon pathology will helpdevelop specific therapeutic agents that preserve axon integrity andenhance regeneration.

During the development of the nervous system, axons respond toextracellular signals that promote the growth as well as those thatinhibit their growth. Some extracellular cues attract axons to growtowards higher concentration and others repel axon away from higherconcentration. The signaling pathways that regulate these opposite axonresponses have profound effect on the extension and removal of axons,although their functions in mature axons have not been wellcharacterized. Studies suggest that axon guidance molecules may play arole in neurological/neurodegenerative disorders, such as amyotrophiclateral sclerosis (ALS).

Atypical protein kinases C (aPKC), including PKCζ and PKCι/λ, playcrucial roles in many cellular processes including cell polarization andsurvival. In neurons, aPKC has been involved in cell polarity (7-13),neurite differentiation (11-13) and axon guidance (14) (15). aPKCmediates axon attraction to Wnts and anterior-posterior axon guidance ofcommissural axons via Wnt-Frizzled3 signaling (14) (15). Through itsinteraction with Par6 and Par3, aPKC is required for axon specificationof hippocampal neurons by regulating the activity of the microtubuleaffinity regulating kinase MARK2 on microtubule-associated protein Tauphosphorylation and microtubule assembly (9). During neuronalpolarization, aPKC is regulated by dishevelled (Dvl) which mediates Wntsignaling (11). aPKC has been also involved in prosurvival signaling inmany different cell types including neural cells (16-23). However, themechanisms underlying the prosurvival function of aPKC in neurons havenot been elucidated. The present disclosure demonstrates that inhibitingaPKC using dominant negative constructs or a myristoylated atypical PKCpseudosubstrate promoted axonal degeneration and neuronal apoptosis.These biochemical studies showed that inhibition of aPKC led tomicrotubule destabilization by increasing MARK2 activity and Tauphosphorylation, resulting in axon degeneration, and eventually neuronalcell body death through the activation of the JNK-cJun pathway. This isthe same signaling mechanism of how aPKC/Par6/Par3 is involved inneuronal polarity and promotes axon elongation (9). Together, theseresults indicate that aPKC is required for both axon elongation duringinitial axon development and maintenance by promoting microtubuleassembly and stability once axons have formed.

Ryk is an atypical receptor tyrosine kinase that binds Wnts.Interestingly, Ryk mediates axon repulsion during development (24-27)and inhibits axon plasticity in adulthood after traumatic injury(28-33). Blocking Ryk signaling into injured dorsal spinal cord preventsaxon retraction and can promote axon regrowth (32). Recently, it wasfound that Ryk expression was increased in motor neurons and axons ofthe ventral spinal cord in a mouse model of ALS, at early stage of thedisease progression, suggesting that Ryk may be involved in early eventsthat trigger neurodegeneration in ALS (6). In this study, it was firsttested whether Ryk regulates aPKC. The findings described herein showthat aPKC activity was increased in Ryk KO neurons, suggesting that Rykmight normally inhibit aPKC. This is a novel discovery of theinteraction between Ryk and aPKC, which may also be relevant tounderstand how Ryk mediates axon repulsion.

It was then determined whether blocking Ryk signaling might preventaxonal degeneration induced by aPKC inhibition and neuronal death in Rykknock-out (KO) mice was analyzed. These studies indicate that inhibitingWnt/Ryk signaling with specific antibodies or by Ryk KO decreasedaPKC-induced axonal degeneration. Because both Ryk and Frizzled3 KO micedie at birth, brain areas that are undergoing neuronal death beforebirth were explored. It was found that an area of cortex shows clearevidence of neuronal death detectable by aCasp3 staining at E18.5, theretrosplenial cortex (RSP), localized between the neocortex andhippocampus.

Consistent with the proposed role of Ryk in promoting degeneration,apoptosis was decreased in E18.5 Ryk KO embryos. Furthermore, it wasfound that apoptosis was greatly increased in Frizzled3 KO embryos inthe RSP at E18.5 and this increase was significantly attenuated inRyk^(+/−) Frizzled3^(−/−) embryos, revealing a genetic interactionbetween Frizzled3 and Ryk. It should be noted that there are 10Frizzleds in the mouse genome. The fact that only the RSP showed clearincrease of aCasp3 immunoreactivity suggests significant functionalredundancy among the Frizzled family members in cortical neuronalprotection at this stage. It cannot be exclude that other areas of thebrain would show increased cell death at later time points, includingneonatal stage. In Frizzled3 KO embryos, only the RSP region of thecortex showed strong increase of death (5 fold), further suggesting thisarea being the most vulnerable before birth. Interestingly, when Ryk wasreduced by half, the increase of death was significantly reduced.

This genetic interaction between the two opposite Wnt receptors whichregulate aPKC in opposite ways further underlie the important role ofaPKC and the complex function of Wnts in regulating neuronal survival.This is also consistent with a recent study showing that Frizzled3 isrequired for spinal cord motor axon survival after initial axonoutgrowth (52). Another recent study also shows that planar cellpolarity signaling pathway mediates axon guidance and aPKC is involvedin amplifying planar cell polarity signaling in growth cone guidance(53) (15). Studies show that Ryk inhibits planar cell polarity signaling(54) (55). Here, the data shows that Ryk and aPKC play opposite roles inmediating axon and neuronal survival. These findings, therefore, willhelp untangle the intricate molecular signaling pathways that thenervous system uses to carefully assemble neural circuits and thedisruption of which may underlie neurological/neurodegenerativedisorders.

Method for Inhibiting Neuron Degeneration

Accordingly, the present invention provides methods and compositions formodulating growth of a nerve cell by contacting the neuron with anagent, thereby inhibiting degeneration of a neuron. In variousembodiments, the agent may be an anti-Ryk monoclonal antibody orantibody fragment that specifically binds to a binding domain of Wntaffecting a Wnt signaling pathway. These methods and compositions can beused in a wide variety of therapeutic contexts where nerve growth andregeneration would be beneficial. For example, an anti-Ryk antibody orantibody fragment affecting a Wnt signaling pathway can be used tostimulate axonal growth of a damaged neuron along the A-P axis of apatient with SCI. Because it has also been observed that the Wnts areexpressed in the several regions in the brain and the components of theWnt signaling pathways are also present in axons of other centralnervous system neurons, it is possible that the anti-Ryk antibody orantibody fragments described herein can be used to modulate growth anddirectional guidance of axons in the central nervous system.

In some embodiments, the methods as described herein result in at leasta 10% decrease (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% decrease) in thedegeneration of a population of neurons or in the degeneration of axonsor cell bodies or dendrites of a neuron in a population of neurons ascompared to a control population of neurons. In some embodiments, themethods as described herein result at least a 10% decrease (e.g., atleast 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or even 100% decrease) in the number of neurons (orneuron bodies, axons, or dendrites thereof) that degenerate in a subjectcompared to the number of neurons (or neuron bodies, axons, or dendritesthereof) that degenerate in a subject that is not administered the oneor more of the agents described herein. In some embodiments, the methodsas described herein result in at least a 10% decrease (e.g., at least15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or even 100% decrease) in one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, or 9) symptoms of a neurological/neurodegenerative disease ordisorder and/or condition. In some embodiments, the methods as describedherein result in at least a 10% decrease (e.g., at least 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, oreven 100% decrease) in the likelihood of developing aneurological/neurodegenerative disease or disorder and/or condition.

The methods of inhibiting neuron degeneration include in vitro, in vivo,and/or ex vivo methods. In some embodiments, the methods are practicedin vivo, i.e., the agent inhibiting neuron degeneration is administeredto a subject. In some embodiments, the methods are practiced ex vivo,i.e., neurons to be treated form part of a nerve graft or a nervetransplant in a subject. In some embodiments, the methods are practicedin vitro.

The methods of inhibiting neuron degeneration can be used to inhibit orprevent neuron degeneration in patients newly diagnosed as having aneurological/neurodegenerative disease or disorder or at risk ofdeveloping a new neurological/neurodegenerative disease or disorder. Onthe other hand, the methods of inhibiting neuron degeneration can alsobe used to inhibit or prevent further neuron degeneration in patientswho are already suffering from, or have symptoms of, aneurological/neurodegenerative disease or disorder. Preventing neurondegeneration includes decreasing or inhibiting neuron degeneration,which may be characterized by complete or partial inhibition of neurondegeneration. This can be assessed, for example, by analysis ofneurological function.

Spinal Cord Injury (SCI)

A large proportion of spinal cord injury patients have incompletelesions, where parts of the spinal cord tissues remain intact. Due tothe strong inhibitory environment in the injured adult spinal cord,especially in the glial scar, and reduced growth potential of adultaxons, the original connections are usually not restored. Nonetheless,the complex circuitry can undergo remodeling to achieve variable levelsof functional recovery with rehabilitative training.

Functional restoration of the corticospinal motor system after spinalcord injury is of principal importance since it is essential forrecovery of voluntary motor control. In rodents, however, the role ofthe corticospinal tract (CST) is more limited, with little effect onlocomotion and hindlimb usage. Nevertheless, the CST is crucial forskilled forelimb motor control. Fine motor skills are lost after adorsal column lesion of the main CST, with a varying extent ofspontaneous recovery.

In order to understand how neural circuits reorganize to regain functionafter injury, functional, anatomical and behavioral analyses wereperformed. The present disclosure demonstrates that the motor cortexremaps such that the cortical areas are no longer used for the hindlimbare recruited to control the forelimb to achieve functional recoveryafter a dorsal column lesion and this reorganization requires continuedtraining. The present disclosure also shows that removing Ryk, areceptor to axon guidance cues Wnts, results in greater CST axonplasticity and cortical circuit remodeling in conjunction withrehabilitative training, leading to maximal functional restoration. Themore gradual and persistent changes in cortical control maps observed inRyk conditional knockout injured mice are likely due to the enhancedchanges in connectivity within the spinal cord and cortical circuitsthat occur in the absence of Wnt-Ryk signaling. It was previously foundthat Wnt-Ryk signaling controls topographic map formation in thedeveloping visual system. The present disclosure reveals a novelfunction of Wnt-Ryk signaling in controlling motor cortex remappingafter spinal cord injury in adulthood.

Previous work has demonstrated that the Wnt signaling, which regulatesaxon guidance in development, has a profound effect on axon plasticityafter injury in the adult spinal cord. The motor output map wascharacterized after spinal cord injury and therefore forelimb motor mapsspread into adjacent regions affected by the injury. It was observedthat an expansion of flexor control area caudally and medially towardscortical regions originally responsible for hindlimb movements (FIGS.7A, 7B, 13A and 13B). These changes are likely stereotypical, becausewrist flexor representations exhibited a medial shift as they recovered,similar to observed shifts of digit representations in primates (FIGS.7A, 13A and 13B).

To specifically test the function of Wnt-Ryk signaling in neurons, aconditional allele of Ryk was generated encoding a repulsive Wntreceptor, motor cortex specific knockout was performed and then thedorsal columns at cervical level 5 (CS) were lesioned. Following Rykconditional knockout, mice recovered on a skilled forelimb reaching taskto 81±7% of peak pre-injury levels at 12 weeks after dorsal columnlesion, compared to only 60±5% in wild type control mice. Thisadditional recovery depends on the segment of the main CST immediatelyrostral to the lesion (C3-CS), as a second dorsal column lesion at C3reduces the functional recovery to control levels. Anatomical analysesshowed significantly increased collateral sprouting of CST above andbelow the CS injury and with pre-synaptic puncta in these axon sprouts.

Using an optogenetic approach, the output map of the motor cortex wasmonitored. It was found that immediately after CS dorsal column lesion,forelimb elbow flexion can be activated by a much larger cortical area,whereas forelimb extension was lost. Over time, the area that activatesforelimb flexion reduced back to the original size and a new area, whichused to activate the hind limb, was recruited to activate forelimbextension. After the second lesion at C3, the control of forelimbflexion was lost but that of the new control of the forelimb extensionwas largely unaffected. In Ryk cKO, these changes are more gradual andpersistent. Finally, mice that did not undergo weekly behavioral testingdisplayed only limited skilled forelimb recovery with performancesimilar to that of mice tested at one week after injury. In the absenceof weekly testing, refinement of cortical motor maps was also impaired,irrespective of Ryk conditional deletion, highlighting the importance oftargeted plasticity.

Alterations of motor output maps have been noted in spinal cord injurypatients for many years but the neural circuit mechanisms remainunknown. It is known that naive transected hindlimb-projectingcorticospinal neurons sprout into cervical spinal cord as early as oneweek post spinal cord injury. However, it is unlikely that earlyexpansion of motor maps above the level of the injury, which wasobserved only 3 days later, was due to sprouting and establishment ofnew connectivity patterns; rather, without being bound by theory, it islikely due to a loss of lateral inhibition within the cortex. However,by 4 weeks after spinal cord injury cortical maps likely reflect theoutput to the remodeled corticospinal circuitry in the cervical spinalcord. It was observed that the greater CST axon collateral numbersinduced following Ryk deletion leads to slight increase of connectionswith motor units distal to the injury site but robust increase withthose rostral to the injury (FIGS. 2H and 3D). Therefore, the changes inconnectivity above the level of injury could be the main source for thechanges of cortical maps (FIG. 7F). For example, the initial expansionof the biceps at 4 weeks post-injury, and subsequent reduction at 8weeks, may result from an initial sprouting of CST collaterals thatnormally project to forelimb motor units in the cervical spinal cord,followed by a subsequent pruning through Hebbian competition. Therecruitment of the hindlimb cortical areas for triceps control mayresult from de novo connections from corticospinal neurons thatoriginally controlled the hindlimb to the forelimb motor units of thecervical spinal cord. These sprouts may either directly contact motorunits or form relays using propriospinal neurons. Conditional deletionof Ryk in corticospinal neurons enhances collateral sprouts and thusrecruits more spinal cord circuitry, a process that likely underliesgreater recovery of voluntary skilled forelimb control. The results ofthe antibody infusion experiments in the spinal cord suggest thatcircuit remodeling in the cervical spinal cord is sufficient to promotefunctional recovery. However, it is plausible that connectivity changeswithin the primary motor cortex also contribute to the remodeling of theentire circuit.

Other descending pathways are also involved in fine motor control andcan partially compensate for the loss of CST input on a skilled forelimbreach task. Additionally, animals with incomplete lesion of the pyramidshave been shown to exhibit similar success rates of skilled forelimbreach to intact control animals through compensatory forelimb movements,indicating that a small proportion of spared CST is capable of restoringfull, if altered, function on the skilled forelimb reach task. In 59% ofthe Ryk cKO mice and 100% of Ryk monoclonal antibody-infused animals,recovery of skilled forelimb reach to levels at or above peak pre-injurylevels was observed. This full recovery clearly requires the novel CSTconnections rostral to the lesion, as a second C3 lesion abolishes theenhanced recovery. While the CST is not the sole component mediatingcontrol of skilled forelimb reach, it is required for the recoveredfunction as pyramidotomy in the mouse model described herein completelyabolished the reaching and grasping behavior. These results suggest thatrestoring at least some CST function is a critical component in recoveryof motor control after injury as compensatory plasticity of other tractsdrives limited recovery in rodents, which are less dependent upon theCST for motor control than primates.

The present disclosure also demonstrates that a Ryk monoclonal antibodycan be a therapeutic tool as blocking Ryk function after lesion leads toimproved functional recovery. It has been shown here that maximalrecovery of the forelimb can be achieved by combining targetedplasticity for the forelimb function (continued reaching and graspingtraining) and molecular manipulation. Therefore, it is anticipated thatcombining targeted plasticity of other functions with molecularmanipulation may allow recovery of other motor or sensory functions. Alarge proportion of patients have incomplete spinal cord injuries,providing a substrate for recovery. This disclosure illustrates thatpromoting circuit plasticity is a promising approach to restore maximalfunction following incomplete spinal cord injury.

Wnt Peptides

Wnts are secreted cysteine-rich glycosylated proteins that play a rolein the development of a wide range of organisms. Wnts are thought tofunction in a variety of developmental and physiological processes sincemany diverse species have multiple conserved Wnt genes (McMahon, 1992;Nusse and Varmus, 1992). The Wnt growth factor family includes at least19 genes identified in mammals, including Wnt1, Wnt2, Wnt2b, Wnt3,Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt 6, Wnt7a, Wnt7b, Wnt8Wnt8b, Wnt9a, Wnt9b,Wnt1 0a, Wnt1 0b, Wnt11, and Wnt16. Similar numbers of Wnt genes arepresent in other vertebrate species (see, e.g., US Pub. No.2011/0065645, incorporated herein by reference in its entirety). Ofcourse, further Wnts may be discovered and/or characterized in thefuture, and those of skill will be able to employ any such Wnts in thecontext of the invention. Further, those of skill will be able to usethe teachings herein to obtain and use Wnts of any species in thecontext of the invention.

Anti-Ryk Antibodies

Antibodies of the invention can be administered by any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapulmonary,intranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, and subcutaneous administration. Inaddition, antibodies can be administered by pulse infusion, particularlywith declining doses of the antibody. Dosing can be by any suitableroute, e.g., by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic.

As used herein, the term “antibody” is used in its broadest sense toinclude polyclonal and monoclonal antibodies, as well as antigen bindingfragments of such antibodies. Antibodies are characterized, in part, inthat they specifically bind to an antigen, particularly to one or moreepitopes of an antigen. The term “binds specifically” or “specificbinding activity” or the like, when used in reference to an antibody,means that an interaction of the antibody and a particular epitope has adissociation constant of at least about 1×10⁻⁶ M, generally at leastabout 1×10⁻⁷ M, usually at least about 1×10⁻⁸M, and particularly atleast about 1×10⁻⁹ M or 1×10⁻¹⁰ M or less. As such, Fab, F(ab′)₂, Fd andFv fragments of an antibody that retain specific binding activity areincluded within the definition of an antibody.

The term “antibody” as used herein includes naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains (see Huse et al., Science246:1275-1281, 1989, which is incorporated herein by reference). Theseand other methods of making, for example, chimeric, humanized,CDR-grafted, single chain, and bifunctional antibodies are well known(Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature341:544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual(Cold Spring Harbor Laboratory Press, 1999); Hilyard et al., ProteinEngineering: A practical approach (IRL Press 1992); Borrabeck, AntibodyEngineering, 2d ed. (Oxford University Press 1995); each of which isincorporated herein by reference). In addition, modified or derivatizedantibodies, or antigen binding fragments of antibodies, such aspegylated (polyethylene glycol modified) antibodies, can be useful forthe present methods.

Antibodies can be tested for anti-target polypeptide activity using avariety of methods well-known in the art. Various techniques may be usedfor screening to identify antibodies having the desired specificity,including various immunoassays, such as enzyme-linked immunosorbentassays (ELISAs), including direct and ligand-capture ELISAs,radioimmunoassays (RIAs), immunoblotting, and fluorescent activated cellsorting (FACS). Numerous protocols for competitive binding orimmunoradiometric assays, using either polyclonal or monoclonalantibodies with established specificities, are well known in the art.Such immunoassays typically involve the measurement of complex formationbetween the target polypeptide and a specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on the target polypeptide is preferred, butother assays, such as a competitive binding assay, may also be employed.See, e.g., Maddox et al, 1983, J. Exp. Med. 158:1211.

The location of the binding target of an antibody used in the inventioncan be taken into consideration in preparation and administration of theantibody. When the binding target is an intracellular molecule, certainembodiments of the invention provide for the antibody or antigen-bindingfragment thereof to be introduced into the cell where the binding targetis located. In one embodiment, an antibody of the invention can beexpressed intracellularly as an intrabody. The term “intrabody,” as usedherein, refers to an antibody or antigen-binding portion thereof that isexpressed intracellularly and that is capable of selectively binding toa target molecule, as described in Marasco, Gene Therapy 4:11-15, 1997;Kontermann, Methods 34:163-170, 2004; U.S. Pat. Nos. 6,004,940 and6,329,173; U.S. Patent Application Publication No. 2003/0104402, and PCTPublication No. WO 03/077945. Intracellular expression of an intrabodyis effected by introducing a nucleic acid encoding the desired antibodyor antigen-binding portion thereof (lacking the wild-type leadersequence and secretory signals normally associated with the geneencoding that antibody or antigen-binding fragment) into a target cell.Any standard method of introducing nucleic acids into a cell may beused, including, but not limited to, microinjection, ballisticinjection, electroporation, calcium phosphate precipitation, liposomes,and transfection with retroviral, adenoviral, adeno-associated viral andvaccinia vectors carrying the nucleic acid of interest.

In another embodiment, internalizing antibodies are provided. Antibodiescan possess certain characteristics that enhance delivery of antibodiesinto cells, or can be modified to possess such characteristics.Techniques for achieving this are known in the art. For example,cationization of an antibody is known to facilitate its uptake intocells (see, e.g., U.S. Pat. No. 6,703,019). Lipofections or liposomescan also be used to deliver the antibody into cells. Where antibodyfragments are used, the smallest inhibitory fragment that specificallybinds to the binding domain of the target protein is generallyadvantageous. For example, based upon the variable-region sequences ofan antibody, peptide molecules can be designed that retain the abilityto bind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology (see, e.g.,Marasco et al., Proc. Natl. Acad. Sci. U.S.A. 90:7889-7893, 1993).

Entry of modulator polypeptides into target cells can be enhanced bymethods known in the art. For example, certain sequences, such as thosederived from HIV Tat or the Antennapedia homeodomain protein are able todirect efficient uptake of heterologous proteins across cell membranes(see, e.g., Chen et al., Proc. Natl. Acad. Sci. U.S.A. 96:4325-4329,1999).

When the binding target is located in the brain, certain embodiments ofthe invention provide for the antibody or antigen-binding fragmentthereof to traverse the blood-brain barrier. Certainneurological/neurodegenerative diseases are associated with an increasein permeability of the blood-brain barrier, such that the antibody orantigen-binding fragment can be readily introduced to the brain. Whenthe blood-brain barrier remains intact, several art-known approachesexist for transporting molecules across it, including, but not limitedto, physical methods, lipid-based methods, and receptor andchannel-based methods.

Physical methods of transporting the antibody or antigen-bindingfragment across the blood-brain barrier include, but are not limited to,circumventing the blood-brain barrier entirely, or by creating openingsin the blood-brain barrier. Circumvention methods include, but are notlimited to, direct injection into the brain (see, e.g., Papanastassiouet al., Gene Therapy 9:398-406, 2002), interstitialinfusion/convection-enhanced delivery (see, e.g., Bobo et al., Proc.Natl. Acad. Sci. U.S.A. 91:2076-2080, 1994), and implanting a deliverydevice in the brain (see, e.g., Gill et al., Nature Med. 9:589-595,2003; and Gliadel Wafers™, Guildford Pharmaceutical). Methods ofcreating openings in the barrier include, but are not limited to,ultrasound (see, e.g., U.S. Pub. No. 2002/0038086), osmotic pressure(e.g., by administration of hypertonic mannitol (Neuwelt, E. A.,Implication of the Blood-Brain Barrier and its Manipulation, Volumes 1and 2, Plenum Press, N.Y., 1989)), permeabilization by, e.g., bradykininor permeabilizer A-7 (see, e.g., U.S. Pat. Nos. 5,112,596, 5,268,164,5,506,206, and 5,686,416), and transfection of neurons that straddle theblood-brain barrier with vectors containing genes encoding the antibodyor antigen-binding fragment (see, e.g., U.S. Pub. No. 2003/0083299).

Lipid-based methods of transporting the antibody or antigen-bindingfragment across the blood-brain barrier include, but are not limited to,encapsulating the antibody or antigen-binding fragment in liposomes thatare coupled to antibody binding fragments that bind to receptors on thevascular endothelium of the blood-brain barrier (see, e.g., U.S. Pub.No. 2002/0025313), and coating the antibody or antigen-binding fragmentin low-density lipoprotein particles (see, e.g., U.S. Pub. No.2004/0204354) or apolipoprotein E (see, e.g., U.S. Pub. No.2004/0131692).

Receptor and channel-based methods of transporting the antibody orantigen-binding fragment across the blood-brain barrier include, but arenot limited to, using glucocorticoid blockers to increase permeabilityof the blood-brain barrier (see, e.g., U.S. Pub. Nos. 2002/0065259,2003/0162695, and 2005/0124533); activating potassium channels (see,e.g., U.S. Pub. No. 2005/0089473), inhibiting ABC drug transporters(see, e.g., U.S. Pub. No. 2003/0073713); coating antibodies with atransferrin and modulating activity of the one or more transferrinreceptors (see, e.g., U.S. Pub. No. 2003/0129186), and cationizing theantibodies (see, e.g., U.S. Pat. No. 5,004,697).

Antibody compositions used in the methods of the invention areformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The antibody need not be, but isoptionally formulated with one or more agents currently used to preventor treat the disorder in question. The effective amount of such otheragents depends on the amount of antibodies of the invention present inthe formulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody (when used alone or in combination with other agents) willdepend on the type of disease to be treated, the type of antibody, theseverity and course of the disease, whether the antibody is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments. Depending on thetype and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1mg/kg-10 mg/kg) of antibody can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the antibody would be in the range fromabout 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof)may be administered to the patient. Such doses may be administeredintermittently, e.g., every week or every three weeks (e.g., such thatthe patient receives from about two to about twenty, or, e.g., about sixdoses of the antibody). An initial higher loading dose, followed by oneor more lower doses may be administered. An exemplary dosing regimencomprises administering an initial loading dose of about 4 mg/kg,followed by a weekly maintenance dose of about 2 mg/kg of the antibody.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

In some embodiments, different antibody regions are illustrated byreference to IgG, which contains four amino acid chains—two longerlength heavy chains and two shorter light chains that areinter-connected by disulfide bonds. The heavy and light chains eachcontain a constant region and a variable region. A heavy chain iscomprised of a heavy chain variable region and a heavy chain constantregion. A light chain is comprised of a light chain variable region anda light chain constant region. In various embodiments, there are threehypervariable regions within the variable regions that are responsiblefor antigen specificity. In various embodiments, the hypervariableregions are referred to as complementarity determining regions (CDR) andare interposed between more conserved flanking regions referred to asframework regions (FW). In various embodiments, the variable regions ofthe heavy and light chains contain a binding domain that interacts withan antigen.

Accordingly, in one aspect, the invention provides an anti-Ryk antibodyand functional fragments thereof that inhibit Wnt-Ryk signaling. Invarious embodiments, the antibody is an isolated monoclonal antibodythat specifically binds to a binding domain of Wnt to inhibit Wnt-Ryksignaling. Sequence data of the key regions of antibodies of theinvention are shown in Tables 2 and 3:

TABLE 2 Sequence Data of Ab5.5 SEQ ID NO: DESCRIPTION SEQUENCE 1CDR of Ab5.5 QDINSY antibody Light Chain Variable Region 2 CDR of Ab5.5RAN antibody Light Chain Variable Region 3 CDR of Ab5.5 LQYDEFPLTantibody Light Chain Variable Region 4 Ab5.5 antibodyDIKMTQSPSSMYASLGERVTITCKASQDINSYLS Light ChainWIQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSG Variable RegionQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGT KLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKD STYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 5 CDR of Ab5.5 GFTFSSYT antibody Heavy Chain Variable Region 6CDR of Ab5.5 ISNGGGGT antibody Heavy Chain Variable Region 7CDR of Ab5.5 HGDNGDYWGHGSTLTVSSAK antibody Heavy Chain Variable Region 8Ab5.5 antibody EVKLVESGGDLVQPGGSLKLSCAASGFTFSSYTM Heavy ChainSWIRQTPEKRLEWVAYISNGGGGTYYPDTVKGR Variable RegionFTISRDNAKNTLYLQMNSLKSEDTAMYYCTRHG DNGDYWGHGSTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGV HTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFI FPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNST FRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTC MITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLH NHHTEKSLSHSPGK

TABLE 3 Sequence Data of Ab11.4 SEQ ID NO: DESCRIPTION SEQUENCE  9CDR of Ab11.4 QDINRY antibody Light Chain Variable Region  2CDR of Ab11.4 RAN antibody Light Chain Variable Region  3 CDR of Ab11.4LQYDEFPLT antibody Light Chain Variable Region 10 Ab11.4DIKMTQSPSSMYASLGERVTITCKASQDINRYLS antibody LightWFQQKPGKSPETLIYRANRLVDGVPSRFSGSGSG Chain VariableQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGT RegionKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLN NFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI VKSFNRNEC  5 CDR of Ab11.4 GFTFSSYTantibody Heavy Chain Variable Region 11 CDR of Ab11.4 ISTGGGSTantibody Heavy Chain Variable Region 12 CDR of Ab11.4HGEFNYWGQGTLVTVSAAK antibody Heavy Chain Variable Region 13 Ab11.4EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTM antibody HeavySWVRQTPEKRLEWVAYISTGGGSTYYPDTVKGR Chain VariableFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHG RegionEFNYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQ TNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVA HPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSW FVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKA PQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNV QKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSP GK

Accordingly, the invention provides an antibody that specifically bindsto a binding domain of Wnt to inhibit Wnt-Ryk signaling. Thus, invarious embodiments, the anti-Ryk antibodies of the present inventioninclude any polypeptide or protein having a binding domain which is, oris substantially identical to the set of CDRs within an antibodyvariable region described herein that is specific for binding to a Wntbinding domain (e.g., at least about 80% identical, at least about 85%identical, at least about 90% identical, at least about 95% identical,or at least 99% identical to those herein). In particular, the antibodyhas a light chain variable region having the CDR sequences set forth inSEQ ID NOs: 1-3 or SEQ ID NOs: 9, 2 and 3; and a heavy chain variableregion having the CDR sequences set forth in SEQ ID Nos: 5-7 or SEQ IDNOs: 5, 11, and 12.

In various embodiments, the light chain variable region has an aminoacid sequence with at least 85% sequence identity to SEQ ID NOs: 4 or10, optionally at least 90% sequence identity to SEQ ID NOs: 4 or 10. Infurther embodiments, the light chain variable region has an amino acidsequence having at least 95% sequence identity to SEQ ID NOs: 4 or 10,optionally at least 99% sequence identity to SEQ ID NOs: 4 or 10. Infurther embodiments, the anti-Ryk antibody Ab5.5 has a light chainvariable region with the amino acid sequence set forth in SEQ ID NO: 4.In further embodiments, the anti-Ryk antibody Ab11.4 has a light chainvariable region with the amino acid sequence set forth in SEQ ID NO: 10.

In various embodiments, the heavy chain variable region has an aminoacid sequence with at least 85% sequence identity to SEQ ID NOs: 8 or13, optionally at least 90% sequence identity to SEQ ID NOs: 8 or 13. Infurther embodiments, the heavy chain variable region has an amino acidsequence having at least 95% sequence identity to SEQ ID NOs: 8 or 13,optionally at least 99% sequence identity to SEQ ID NOs: 8 or 13. Infurther embodiments, the anti-Ryk antibody Ab5.5 has a heavy chainvariable region with the amino acid sequence set forth in SEQ ID NO: 8.In further embodiments, the anti-Ryk antibody Ab11.4 has a heavy chainvariable region with the amino acid sequence set forth in SEQ ID NO: 13.

While the heavy chain variable region and light chain variable regionmay be combined with other light chain variable regions and other heavychain variable regions respectively, so long as specific binding to abinding domain of Wnt can be maintained to inhibit Wnt-Ryk signalingbinding, in some embodiments, the anti-Ryk antibody includes a heavychain variable region having at least 85% sequence identity to the aminoacid sequence set forth in SEQ ID NO: 8, the heavy chain variable regionfurther having three CDR sequences set forth in SEQ ID NOs: 5-7; and alight chain region variable region having at least 85% sequence identityto the amino acid sequence set forth in SEQ ID NO: 4, the light chainvariable region also having the three CDR sequences set forth in SEQ IDNOs: 1-3. In some embodiments, the anti-Ryk antibody includes a heavychain variable region having at least 85% sequence identity to the aminoacid sequence set forth in SEQ ID NO: 13, the heavy chain variableregion further having three CDR sequences set forth in SEQ ID NOs: 5,11, and 12; and a light chain region variable region having at least 85%sequence identity to the amino acid sequence set forth in SEQ ID NO: 10,the light chain variable region also having the three CDR sequences setforth in SEQ ID NOs: 9, 2, and 3.

In further embodiments, the heavy chain variable region has at least 90%sequence identity to the amino acid sequence of SEQ ID NO: 8 and thelight chain variable region has at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 4. In still further embodiments, theheavy chain variable region has at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 8 and the light chain variable regionhas at least 95% sequence identity to the amino acid sequence of SEQ IDNO: 4. In still further embodiments, the heavy chain variable region hasat least 99% sequence identity to the amino acid sequence of SEQ ID NO:8 and the light chain variable region has at least 99% sequence identityto the amino acid sequence of SEQ ID NO: 4. In the anti-Ryk antibodyAB5.5, the heavy chain variable region has the amino acid sequence ofSEQ ID NO: 8 and the light chain variable region has the amino acidsequence of SEQ ID NO: 4.

In further embodiments, the heavy chain variable region has at least 90%sequence identity to the amino acid sequence of SEQ ID NO: 13 and thelight chain variable region has at least 90% sequence identity to theamino acid sequence of SEQ ID NO: 10. In still further embodiments, theheavy chain variable region has at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 13 and the light chain variable regionhas at least 95% sequence identity to the amino acid sequence of SEQ IDNO: 10. In still further embodiments, the heavy chain variable regionhas at least 99% sequence identity to the amino acid sequence of SEQ IDNO: 13 and the light chain variable region has at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO: 10. In the anti-Rykantibody AB11.4, the heavy chain variable region has the amino acidsequence of SEQ ID NO: 13 and the light chain variable region has theamino acid sequence of SEQ ID NO: 10.

As indicated above, the invention encompasses variations in sequencearound the CDRs of the antibody within a percent sequence identity. Theterm “percent sequence identity”, with respect to two amino acid orpolynucleotide sequences, refers to the percentage of residues that areidentical in the two sequences when the sequences are optimally aligned.Thus, 80% amino acid sequence identity means that 80% of the amino acidsin two optimally aligned polypeptide sequences are identical. Likewise,85% amino acid sequence identity means that 85% of the amino acids intwo optimally aligned polypeptide sequences are identical; and 90%, 95%,and 99% amino acid sequence identity means that 90%, 95%, and 99%respectively of the amino acids in two optimally aligned polypeptidesequences are identical.

Variations in sequence identity are permitted outside of the CDRsbecause not all amino acid residues within the heavy and light variableregions are required for binding Ryk at a binding domain of Wnt. Inparticular regions outside of CDRs, such as framework regions, may bemutated without losing Ryk binding capability. Still further, frameworkregions may be further mutated and thus vary in sequence when adaptingthe CDRs for the treatment of different species. Mutations or variationscan be described by use of the following nomenclature: position (#);substituted amino acid residue(s). According to this nomenclature, thesubstitution of, for instance, an alanine residue for a glycine residueat position can be indicated as A#G, where # represents the position.When an amino acid residue at a given position is substituted with twoor more alternative amino acid residues, these residues are separated bya comma or a slash. For example, substitution of alanine with eitherglycine or glutamic acid can be indicated as #G/E, or #G, #E. Thedeletion of alanine in the same position can be shown as Ala#* or A#* or*#Ala or *#A, where # refers to the position of the amino acid. Multiplemutations are separated by a plus sign or a slash. For example, twomutations in positions (each indicated by “#”) substituting alanine andglutamic acid for glycine and serine, respectively, are indicated asA#G+E#S or A#G/E#S. When an amino acid residue at a given position # issubstituted with two or more alternative amino acid residues, theseresidues are separated by a comma or a slash. For example, substitutionof alanine at a position # with either glycine or glutamic acid isindicated as A#G,E or A#G/E, or A#G, A#E. When a position # suitable formodification is identified herein without any specific modificationbeing suggested, it is to be understood that any amino acid residue maybe substituted for the amino acid residue present in the position. Thus,for instance, when a modification of an alanine at a position # ismentioned but not specified, it is to be understood that the alanine maybe deleted or substituted for any other amino acid residue (i.e., anyone of R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V).

Referring back to SEQ ID NOs: 1-13, the invention can be defined as aset of CDR regions with anti-Ryk binding activity; however, in preferredembodiments the CDR peptides (each corresponding to a single CDR) arejoined to form an anti-Ryk antibody having a variable region forspecific binding to a Wnt binding domain. Again, the antibody variableregion can be present in, for example, a complete antibody, an antibodyfragment (e.g., F(ab), F(ab′)₂, scFv, minibody, tetrabody and others) ora recombinant derivative of an antibody or antibody fragment. In someaspects, the antibody variable region is present in a recombinantderivative. Examples of recombinant derivatives include single-chainantibodies, diabody, triabody, tetrabody, and miniantibody. In someembodiments, an anti-Ryk antibody also contains one or more variableregions recognizing the same or different epitopes.

In various embodiments, the anti-Ryk antibodies of the invention maycontain additional components including, but not limited to, componentsother than variable regions or additional variable regions that provide,or help provide, useful and/or additional activities. Useful activitiesinclude, for example, antibody effector functions such asantibody-dependent cellular cytoxicity, phagocytosis,complement-dependent cytoxicity, and half-life/clearance rate. In someembodiments, antibody effector functions are mediated by different hostcomponents, such as Fcγ receptors, neonatal Fc receptor (FcRn), and C1q.In various embodiments, different types of antibody components oralterations are used to enhance effector functions. Examples of usefulcomponents or alternations include the use of non-fucosylatedoligosaccharides, amino acids altered to have increased stability, aminoacids with enhanced binding to FcRn, amino acid alterations withenhanced binding to a Fcγ receptor, and amino acid alterations withdecreased binding affinity to a Fcγ receptor.

In various embodiments, the anti-Ryk antibodies of the invention maycontain additional components to alter the physiochemical properties ofthe protein, providing pharmacological advantages. For example, theattachment of polyethylene glycol (“PEG”) to molecules, in someembodiments, improves safety by reducing toxicity and increasingefficiency of the molecules when used as therapeutics. Physiochemicalalterations include, but are not limited to, changes in conformation,electrostatic binding, and hydrophobicity which can work together toincrease systemic retention of a therapeutic agent. Additionally, byincreasing the molecular weight of an anti-Ryk antibody or functionalfragment thereof by attaching a PEG moiety, pharmacological advantagesmay include extended circulating life, increased stability, and enhancedprotection from host proteases. PEG attachment can also influencebinding affinity of the therapeutic moiety to cell receptors. PEG is anon-ionic polymer composed of repeating units (—O—CH₂—CH₂—) to make arange of molecular weight polymers from 400 to greater than 15,000(e.g., PEG polymers with molecular weights of up to 400,000 arecommercially available). Methods for incorporating PEG or long chainpolymers of PEG are well known in the art (described, for example, inVeronese, F. M., et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald,R. B., et al., Adv. Drug Deliv. Rev. 55: 217-50 (2003); Roberts, M. J.,et al., Adv. Drug Deliv. Rev., 54: 459-76 (2002)), the contents of whichis incorporated herein by reference. Other methods of polymerconjugations known in the art can also be used in the present invention.

Thus, the anti-Ryk antibody or functional fragment thereof may bederivatized, linked to or co-expressed with another functional molecule,e.g., another peptide or protein (e.g., a Fab fragment). For example,the antibody can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., to produce abispecific or a multispecific antibody), a cytotoxin, cellular ligand orantigen (e.g., to produce an immunoconjugate, such as an immunotoxin).The antibody also can be linked to other therapeutic moieties, e.g., aradioisotope, a small molecule anti-cancer drug, an anti-inflammatoryagent, or an immunosuppressive agent. Accordingly, the present inventionencompasses a large variety of antibody conjugates, bispecific andmultispecific molecules, and fusion proteins, all of which specificallybind to a binding domain of Wnt or specifically bind to the same epitopeon Wnt as does a reference antibody or antibody fragment, orcross-competes for specific binding to Wnt with a reference antibody orantibody fragment, as described herein.

Nucleic Acids

Nucleic acid sequences encoding the anti-Ryk polypeptide sequences,which include any of SEQ ID NOs: 1-13 are also provided. Recombinantnucleic acids encoding anti-Ryk antibodies are particularly useful forexpression in a host cell that in effect serves as a factory for theanti-Ryk antibodies. In various embodiments, nucleic acids are isolatedwhen purified away from other cellular components or other contaminants(e.g., other nucleic acids or proteins present in the cell) by standardtechniques including, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well-knownin the art. See e.g., F. Ausubel, et al., ed. (1987) Current Protocolsin Molecular Biology, Greene Publishing and Wiley Interscience, NewYork. In various embodiments, a nucleic acid is, for example, DNA or RNAand may or may not contain intronic sequences. In a preferredembodiment, the nucleic acid is a cDNA molecule. In various embodiments,a recombinant nucleic acid provides a recombinant gene encoding theanti-Ryk antibody that exists autonomously from a host cell genome or aspart of the host cell genome.

In some embodiments, a recombinant gene contains nucleic acids encodinga protein along with regulatory elements for protein expression.Generally, the regulatory elements that are present in a recombinantgene include a transcriptional promoter, a ribosome binding site, aterminator, and an optionally present operator. A promoter is defined asa DNA sequence that directs RNA polymerase to bind to DNA and initiateRNA synthesis. Antibody associated introns may also be present. Thedegeneracy of the genetic code is such that, for all but two aminoacids, more than a single codon encodes a particular amino acid. Thisallows for the construction of synthetic DNA that encodes a proteinwhere the nucleotide sequence of the synthetic DNA differs significantlyfrom the nucleotide sequences disclosed herein, but still encodes such aprotein. Such synthetic DNAs are intended to be within the scope of thepresent invention.

Diseases

The anti-Ryk antibodies or antibody fragments described herein can beused in methods for inhibiting neuron (e.g., axon) degeneration. Theseantibodies or antibody fragments are, therefore, useful in the therapyof, for example, (i) disorders of the nervous system (e.g.,neurological/neurodegenerative diseases or disorders), (ii) conditionsof the nervous system that are secondary to a disease, condition, ortherapy having a primary effect outside of the nervous system, (iii)injuries to the nervous system caused by physical, mechanical, orchemical trauma, (iv) pain, (v) ocular-related neurodegeneration, (vi)memory loss, and (vii) psychiatric disorders. Non-limiting examples ofsome of these diseases, conditions, and injuries are provided below.

Examples of neurological/neurodegenerative diseases and conditions thatcan be prevented or treated according to the invention includeamyotrophic lateral sclerosis (ALS), trigeminal neuralgia,glossopharyngeal neuralgia, Bell's Palsy, myasthenia gravis, musculardystrophy, progressive muscular atrophy, primary lateral sclerosis(PLS), pseudobulbar palsy, progressive bulbar palsy, spinal muscularatrophy, progressive bulbar palsy, inherited muscular atrophy,invertebrate disk syndromes (e.g., herniated, ruptured, and prolapseddisk syndromes), cervical spondylosis, plexus disorders, thoracic outletdestruction syndromes, peripheral neuropathies, prophyria, mildcognitive impairment, Alzheimer's disease, Huntington's disease,Parkinson's disease, Parkinson's-plus diseases (e.g., multiple systematrophy, progressive supranuclear palsy, and corticobasal degeneration),dementia with Lewy bodies, frontotemporal dementia, demyelinatingdiseases (e.g., Guillain-Barre syndrome and multiple sclerosis),Charcot-Marie-Tooth disease (CMT; also known as Hereditary Motor andSensory Neuropathy (HMSN), Hereditary Sensorimotor Neuropathy (HSMN),and Peroneal Muscular Atrophy), prion disease (e.g., Creutzfeldt-Jakobdisease, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familialinsomnia (FFI), and bovine spongiform encephalopathy (BSE, commonlyknown as mad cow disease)), Pick's disease, epilepsy, and AIDS dementialcomplex (also known as HIV dementia, HIV encephalopathy, andHIV-associated dementia).

The methods of the invention can also be used in the prevention andtreatment of ocular-related neurodegeneration and related diseases andconditions, such as glaucoma, lattice dystrophy, retinitis pigmentosa,age-related macular degeneration (AMD), photoreceptor degenerationassociated with wet or dry AMD, other retinal degeneration, optic nervedrusen, optic neuropathy, and optic neuritis. Non-limiting examples ofdifferent types of glaucoma that can be prevented or treated accordingto the invention include primary glaucoma (also known as primaryopen-angle glaucoma, chronic open-angle glaucoma, chronic simpleglaucoma, and glaucoma simplex), low-tension glaucoma, primaryangle-closure glaucoma (also known as primary closed-angle glaucoma,narrow-angle glaucoma, pupil-block glaucoma, and acute congestiveglaucoma), acute angle-closure glaucoma, chronic angle-closure glaucoma,intermittent angle-closure glaucoma, chronic open-angle closureglaucoma, pigmentary glaucoma, exfoliation glaucoma (also known aspseudoexfoliative glaucoma or glaucoma capsulare), developmentalglaucoma (e.g., primary congenital glaucoma and infantile glaucoma),secondary glaucoma (e.g., inflammatory glaucoma (e.g., uveitis and Fuchsheterochromic iridocyclitis)), phacogenic glaucoma (e.g., angle-closureglaucoma with mature cataract, phacoanaphylactic glaucoma secondary torupture of lens capsule, phacolytic glaucoma due to phacotoxic meshworkblockage, and subluxation of lens), glaucoma secondary to intraocularhemorrhage (e.g., hyphema and hemolytic glaucoma, also known aserythroclastic glaucoma), traumatic glaucoma (e.g., angle recessionglaucoma, traumatic recession on anterior chamber angle, postsurgicalglaucoma, aphakic pupillary block, and ciliary block glaucoma),neovascular glaucoma, drug-induced glaucoma (e.g., corticosteroidinduced glaucoma and alpha-chymotrypsin glaucoma), toxic glaucoma, andglaucoma associated with intraocular tumors, retinal detachments, severechemical burns of the eye, and iris atrophy.

Certain diseases and conditions having primary effects outside of thenervous system can lead to damage to the nervous system, which can betreated according to the methods of the present invention. Examples ofsuch conditions include peripheral neuropathy and neuralgia caused by,for example, diabetes, cancer, AIDS, hepatitis, kidney dysfunction,Colorado tick fever, diphtheria, HIV infection, leprosy, lyme disease,polyarteritis nodosa, rheumatoid arthritis, sarcoidosis, Sjogrensyndrome, syphilis, systemic lupus erythematosus, and amyloidosis.

In addition, the methods of the invention can be used in the treatmentof nerve damage, such as peripheral neuropathy, which is caused byexposure to toxic compounds, including heavy metals (e.g., lead,arsenic, and mercury) and industrial solvents, as well as drugsincluding chemotherapeutic agents (e.g., vincristine and cisplatin),dapsone, HIV medications (e.g., Zidovudine, Didanosine, Stavudine,Zalcitabine, Ritonavir, and Amprenavir), cholesterol lowering drugs(e.g., Lovastatin, Indapamid, and Gemfibrozil), heart or blood pressuremedications (e.g., Amiodarone, Hydralazine, Perhexiline), andMetronidazole.

The methods of the invention can also be used to treat injury to thenervous system caused by physical, mechanical, or chemical trauma. Thus,the methods can be used in the treatment of peripheral nerve damagecaused by physical injury (associated with, e.g., burns, wounds,surgery, and accidents), ischemia, prolonged exposure to coldtemperature (e.g., frost-bite), as well as damage to the central nervoussystem due to, e.g., stroke or intracranial hemorrhage (such as cerebralhemorrhage).

Further, the methods of the invention can be used in the prevention ortreatment of memory loss such as, for example, age-related memory loss.Types of memory that can be affected by loss, and thus treated accordingto the invention, include episodic memory, semantic memory, short-termmemory, and long-term memory. Examples of diseases and conditionsassociated with memory loss, which can be treated according to thepresent invention, include mild cognitive impairment, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, chemotherapy,stress, stroke, and traumatic brain injury (e.g., concussion).

The methods of the invention can also be used in the treatment ofpsychiatric disorders including, for example, schizophrenia, delusionaldisorder, schizoaffective disorder, schizopheniform, shared psychoticdisorder, psychosis, paranoid personality disorder, schizoid personalitydisorder, borderline personality disorder, anti-social personalitydisorder, narcissistic personality disorder, obsessive-compulsivedisorder, delirium, dementia, mood disorders, bipolar disorder,depression, stress disorder, panic disorder, agoraphobia, social phobia,post-traumatic stress disorder, anxiety disorder, and impulse controldisorders (e.g., kleptomania, pathological gambling, pyromania, andtrichotillomania).

In addition to the in vivo methods described above, the methods of theinvention can be used to treat nerves ex vivo, which may be helpful inthe context of nerve grafts or nerve transplants. Thus, the compoundsprovided herein can be useful as components of culture media for use inculturing nerve cells in vitro.

The antibodies or antibody fragments described herein can be optionallycombined with or administered in concert with each other or other agentsknown to be useful in the treatment of the relevant disease orcondition. Thus, in the treatment of ALS, for example, the compounds canbe administered in combination with Riluzole (Rilutek), minocycline,insulin-like growth factor 1 (IGF-I), and/or methylcobalamin. In anotherexample, in the treatment of Parkinson's disease, inhibitors can beadministered with L-dopa, dopamine agonists (e.g., bromocriptine,pergolide, pramipexole, ropinirole, cabergoline, apomorphine, andlisuride), dopa decarboxylase inhibitors (e.g., levodopa, benserazide,and carbidopa), and/or MAO-B inhibitors (e.g., selegiline andrasagiline). In a further example, in the treatment of Alzheimer'sdisease, inhibitors can be administered with acetylcholinesteraseinhibitors (e.g., donepezil, galantamine, and rivastigmine) and/or NMDAreceptor antagonists (e.g., memantine). The combination therapies caninvolve concurrent or sequential administration, by the same ordifferent routes, as determined to be appropriate by those of skill inthe art. The invention also includes pharmaceutical compositions andkits including combinations as described herein.

In the context of the invention, the terms “contact” or “contacting” aredefined to mean any manner in which a compound is brought into aposition where it can mediate, modulate, or inhibit the growth of aneuron. “Contacting” can comprise injecting a diffusable ornon-diffusable substance into the neuron or an area adjacent a neuron.“Contacting” can comprise placing a nucleic acid encoding a compoundinto or close to a neuron or non-neuronal cell in a manner such that thenucleic acid is expressed to make the compound in a manner in which itcan act upon the neuron. Those of skill in the art, following theteachings of this specification, will be able to contact neurons withsubstances in any manner.

The methods for modulating growth of a neuron may, in certainembodiments, be methods for stimulating growth of a neuron, methods forregenerating a damaged neuron, or methods for guiding growth of a neuronalong the anterior-posterior axis. In other embodiments, the methods formodulating growth of a neuron are further defined as methods fordirectionally orienting axon growth of a neuron between the spinal cordand the brain.

In certain embodiments, the neuron is contacted with an anti-Rykmonoclonal antibody or antibody fragment that specifically binds to abinding domain of Wnt affecting a Wnt signaling pathway, and may furtherinvolve exposing the neuron to a gradient of the anti-Ryk monoclonalantibody or antibody fragment that specifically binds to a bindingdomain of Wnt affecting a Wnt signaling pathway. The gradient may be inthe spinal cord, such as a decreasing anterior-posterior gradient withinthe spinal cord. In other embodiments, exposing the neuron to thegradient involves stimulating directionally-oriented axon growth of theneuron along the anterior-posterior axis. Any direction of axon growthis contemplated by the present invention. In certain embodiments, theaxon growth is directed from the spinal cord to the brain, such as inthe growth of neurons in ascending somatosensory pathways. In otherembodiments, the axon growth is directed from the brain to the spinalcord, such as in the growth of neurons in descending motor pathways orother regulatory pathways. In further embodiments, the axon growth isdirected along the spinothalamic pathway.

The present invention also includes methods of modulating growth of aneuron in a subject, including: (a) providing a composition thatincludes an anti-Ryk antibody or antibody fragment that specificallybinds to a binding domain of Wnt affecting a Wnt signaling pathway; anda pharmaceutical preparation suitable for delivery to the subject; and(b) administering the composition to the subject. The methods formodulating neuron growth of the present invention contemplatemeasurement of neuronal growth by any known means, as discussed above.For example, the method of modulating neuron growth may be defined as amethod of promoting growth and regeneration of a neuron in a subject, amethod of promoting axon growth and regeneration in a subject, or amethod of promoting directionally-oriented axon growth in a subject.Directionally-oriented axon growth may be along the anterior-posterioraxis such as from the spinal cord to the brain, or from the brain to thespinal cord.

In another aspect, the invention provides a composition comprising theantibody or antibody fragment of the invention, which can be preparedfor administration to a subject by mixing the antibody or immunogenicpeptide fragment with physiologically acceptable carriers or excipients.Such carriers will be nontoxic to recipients at the dosages andconcentrations employed. Ordinarily, the preparation of suchcompositions entails combining the particular antibody with saline,buffers, antioxidants such as ascorbic acid, low molecular weight (lessthan about 10 residues) polypeptides, proteins, amino acids,carbohydrates including glucose or dextrans, or chelating agents such asEDTA, glutathione and other stabilizers and excipients. Suchcompositions can be in suspension, emulsion or lyophilized form and areformulated under conditions such that they are suitably prepared andapproved for use in the desired application.

A physiologically acceptable carrier or excipient can be any materialthat, when combined with an immunogenic peptide or a polynucleotide ofthe invention, allows the ingredient to retain biological activity anddoes not undesirably disrupt a reaction with the subject's immunesystem. Examples include, but are not limited to, any of the standardphysiologically acceptable carriers such as a phosphate buffered salinesolution, water, emulsions such as oil/water emulsion, and various typesof wetting agents. Preferred diluents for aerosol or parenteraladministration are phosphate buffered saline or normal (0.9%) saline.Compositions comprising such carriers are formulated by well-knownconventional methods (see, for example, Remington's PharmaceuticalSciences, Chapter 43, 14th Ed., Mack Publishing Co., Easton Pa. 18042,USA).

For administration to a subject, a peptide, or an encodingpolynucleotide, generally is formulated as a composition. Accordingly,the present invention provides a composition, which generally contains,in addition to the peptide or polynucleotide of the invention, a carrierinto which the peptide or polynucleotide can be conveniently formulatedfor administration. For example, the carrier can be an aqueous solutionsuch as physiologically buffered saline or other solvent or vehicle suchas a glycol, glycerol, an oil such as olive oil or an injectable organicesters. A carrier also can include a physiologically acceptable compoundthat acts, for example, to stabilize the peptide or encodingpolynucleotide or to increase its absorption. Physiologically acceptablecompounds include, for example, carbohydrates, such as glucose, sucroseor dextrans, antioxidants, such as ascorbic acid or glutathione,chelating agents, low molecular weight proteins or other stabilizers orexcipients. Similarly, a cell that has been treated in culture forpurposes of the practicing the methods of the invention, for example,synovial fluid mononuclear cells, dendritic cells, or the like, also canbe formulated in a composition when the cells are to be administered toa subject.

It will be recognized to the skilled clinician that choice of a carrieror excipient, including a physiologically acceptable compound, depends,for example, on the manner in which the peptide or encodingpolynucleotide is to be administered, as well as on the route ofadministration of the composition. Where the composition is administeredunder immunizing conditions, i.e., as a vaccine, it generally isadministered intramuscularly, intradermally, or subcutaneously, but alsocan be administered parenterally such as intravenously, and can beadministered by injection, intubation, or other such method known in theart. Where the desired modulation of the immune system is tolerization,the composition preferably is administered orally, or can beadministered as above.

Screening Methods

In another aspect, the invention provides a method of screening for atherapeutic/test/candidate agent for treating a neurological disease ordisorder. The method includes administering a test agent to thetransgenic non-human mammal described herein and evaluating the effectof the test agent on at least one of: the amount of atypical proteinkinases C (aPKC) or MARK2 protein, the level of aPKC or MARK2 activityor the level of aPKC or MARK2 in at least one disease-relevant tissue ofthe transgenic non-human mammal, wherein at least one of: a decrease inthe amount of aPKC protein, an increase in the amount of MARK2 protein,a decrease in the level of aPKC activity, an increase in the level ofMARK2 activity, a reduction in the level of aPKC, or an increase in thelevel of MARK2 in at least one disease-relevant tissue relative to asimilar transgenic non-human mammal that does not receive the test agentindicates the test agent is therapeutic for the neurological disease ordisorder.

A “test agent” or “candidate agent” refers to an agent that is to bescreened in one or more of the assays described herein. The agent can bevirtually any chemical compound. It can exist as a single isolatedcompound or can be a member of a chemical (e.g., combinatorial) library.In one embodiment, the test agent is a small organic molecule. The termsmall organic molecules refers to molecules of a size comparable tothose organic molecules generally used in pharmaceuticals.

Conventionally, new chemical entities with useful properties aregenerated by identifying a chemical compound (called a “lead compound”)with some desirable property or activity, creating variants of the leadcompound, and evaluating the property and activity of those variantcompounds.

Any agent identified as being a potential therapeutic for treating aneurological disease or disorder may be brought into association with apharmaceutically acceptable carrier, which constitutes one or moreaccessory ingredients. The term “pharmaceutically acceptable,” when usedin reference to a carrier, is meant that the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

Pharmaceutically acceptable carriers useful for formulating an agent foradministration to a subject are well known in the art and include, forexample, aqueous solutions such as water or physiologically bufferedsaline or other solvents or vehicles such as glycols, glycerol, oilssuch as olive oil or injectable organic esters. A pharmaceuticallyacceptable carrier can contain physiologically acceptable compounds thatact, for example, to stabilize or to increase the absorption of theconjugate. Such physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients. Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the physico-chemical characteristics of thetherapeutic agent and on the route of administration of the composition,which can be, for example, orally, intranasally or any other such methodknown in the art. The pharmaceutical composition also can contain asecond (or more) compound(s) such as a diagnostic reagent, nutritionalsubstance, toxin, or therapeutic agent, for example, a cancerchemotherapeutic agent and/or vitamin(s).

The total amount of a compound or composition to be administered inpracticing a method of the invention can be administered to a subject asa single dose, either as a bolus or by infusion over a relatively shortperiod of time, or can be administered using a fractionated treatmentprotocol, in which multiple doses are administered over a prolongedperiod of time. One skilled in the art would know that the amount of theplasma expander used to treat blood loss in a subject depends on manyfactors including the age and general health of the subject as well asthe route of administration and the number of treatments to beadministered. In view of these factors, the skilled artisan would adjustthe particular dose as necessary. In general, the formulation of thepharmaceutical composition and the routes and frequency ofadministration are determined, initially, using Phase I and Phase IIclinical trials.

The following examples are intended to illustrate but not limit theinvention.

Example 1 Methods

Animals—

Time-mated pregnant CD1 mice rats were purchased from Charles River.Ryk-deficient mice were obtained from Steven Stacker (Ludwig Institutefor Cancer Research, Melbourne, VIC, Australia) (50).Frizzled3-deficient and were obtained from Jeremy Nathans (John HopkinsUniversity, Baltimore, Md.) (51). Genotypes were determined by standardPCR. The day of vaginal plug detection was considered E0.5. Animals werehoused in a temperature-controlled room with standard laboratory foodand water provided ad libitum.

Antibodies, Reagents and Plasmidic Constructs—

Myristoylated aPKC and cPKC pseudosubstrates were purchased from EnzoLife Sciences-Biomol (#BML-P-219 and #BML-P205 respectively).

Monoclonal mouse Ryk antibodies were generated against the ectodomain ofRyk, amino-acids 90-183, fused with maltose binding protein.

Commercial primary antibodies used in this study included P-PKCζ T410(Santa Cruz sc-101778, rabbit 1:1000), Total PKCζ (Santa Cruz C-20sc-216, rabbit, 1:2000), P-MARK2 T595 (Abcam, rabbit 1:1000), TotalMARK2 (Abcam, goat 1:1000), SMI-312 (Covance, mouse, 1:1000), MAP2(Abcam, rabbit 1:1500), βIII-tubulin (Covance, mouse, 1:1000),Rhodamine-phalloidin (Invitrogen, 1:100), P-Tau S262 (Invitrogen,rabbit, 1:1000), Tau (Tau5, Millipore, mouse, 1:2000 for cell culture,1:4000 for western blotting), P-JNK/SAPK T183/T185 (Cell Signaling,rabbit, 1:1000), Total JNK/SAPK (Cell Signaling, 1:1000), P-c-Jun S63(Cell Signaling, rabbit 1:1000), Total c-Jun (Cell Signaling, rabbit1:1000), GAPDH (Millipore-Chemicon, mouse, 1:50,000), actin (mouse,1:5000), activated Caspase-3 (aCasp3, Cell signaling, rabbit, 1:1000 oncell cultures, 1:500 on tissue sections) and CTIP2 (Abcam, rat, 1:500).

cDNAs of PKCζ-WT, PKCζ-KD (K281W) and PKCζ-T410A plasmidic constructs(56) were cloned into pCIG2-EGFP plasmid (14). pCIG2-Tau-WT andpCIG2-Tau-S262A plasmids were provided by Georges Mairet-Coello (TheScripps Research Institute, La Jolla, Calif.) (57).

Cerebral Cortical Cell Culture—

E16.5 mouse pregnant females were sacrificed by CO₂ asphyxia andcervical dislocation. Embryos were removed from uterine horns. Skin,skull, and meninges were removed from embryo heads. The cortex wasdissected in L-15 medium (Sigma), digested with 0.025% trypsin/0.221 mMEDTA (Mediatech) for 20 min followed by incubation in 5% horse serumprepared in L-15 medium for 5 min to inhibit trypsin. Cortices weremechanically dissociated and cells were plated at 500 cells/mm² onpoly-D-lysine (33.3 μg/ml, Millipore) and laminin (3.3 μg/ml,Invitrogen)-coated 12 mm glass coverslips in 24-well plates or at 1050cells/mm² on poly-D-lysine (0.1 mg/ml)-coated 6 well plates (35 mmdishes). Cells were incubated in Neurobasal (Gibco) medium supplementedwith B27 (Gibco) containing Glutamax (Gibco), 40 mM Glucose, 100 U/mlpenicillin and 1 mg/ml streptomycin (Mediatech). Cultures weremaintained in a humidified 5% CO₂/air incubator at 37° C.

Magnetofection—

For magnetofection, cortices from E17.5 embryos were dissociated inpapain (Worthington) supplemented with DNAse I (100 mg/ml, Sigma) for 20min at 37° C., washed three times and manually triturated in platingmedium. Cells were plated at 565/mm² on 12 mm-glass bottom dishes coatedwith poly-D-lysine (1 mg/ml, Sigma). Cells were cultured in neurobasalmedium supplemented with 2.5% fetal bovine serum (Gemini), B27 (1×),L-glutamine (2 mM) and penicillin (2.5 U/ml)-streptomycin (2.5 mg/ml)(Invitrogen). Neurons were transfected at DIV3 by magnetofection usingNeuroMag (OZ Bioscience), according to manufacturer's instructions andas previously described (57). Briefly, 2 μg cDNA was incubated withNeuroMag in neurobasal medium for 15 min at room temperature, and thenthe mixture was applied dropwise on culture cells. Cultures were placedon magnet for 20 min for transfection. Cotransfections were performed ata 1:1 ratio (w/w).

Immunohistochemistry, Immunocytochemistry and TUNEL Assay—

Cell cultures were fixed with 4% PFA for 30 min. Embryonic brains weredissected in cold PBS, fixed in 4% PFA for 2 h and incubated overnightin 30% sucrose for cryoprotection. Brains were embedded in a commercialembedding medium (Tissue-Tek, Sakura Finetek), quickly frozen on dry-iceand coronally sectioned at a thickness of 20 μm using acryostat-microtome (Leica). Sections were mounted on Superfrost Plusslides (Thermo Fisher Scientific) and stored at −20° C. Cells orsections were permeabilized in PBS containing 0.3% triton X-100 (PBS-T)for 10 min and incubated with the primary antibody overnight at 4° C.Monoclonal antibodies were diluted in PBS-T and polyclonal antibodieswere diluted in a PBS-T solution containing 10% lactoproteins, 1% bovineserum albumin. Staining was visualized using Alexa Fluor 588(1:500-1:1000), 594 (1:500-1:1000) or 649 (1:200-1:500) conjugatedsecondary antibodies (Jackson ImmunoReasearch). For CTIP2immunodetection, sections were submitted to antigen retrieval procedurein 10 mM citrate buffer pH=6 at 90° C.-95° C. for 10 min. Sections werecounterstained with DAPI (Thermo Fisher Scientific). TUNEL was performedusing the In situ cell death detection kit TMR red (Roche) according tothe manufacturer's instructions.

Western-Blotting—

Cortical cells cultured in 6 well plates were washed twice with PBS (pH7.4), scrapped using a rubber policeman and lysed in lysis buffercontaining 20 μM Tris-HCl pH7.6, 150 μM NaCl, 0.1% SDS, 1% triton X-100,protease inhibitors (cOmplete mini tablets, Roche Applied Science),phosphatase inhibitors (PhosSTOP, Roche Applied Science). Twentymicrograms of proteins were separated by SDS PAGE and thenelectro-transferred onto polyvinylidene difluoride membranes (Bio-Rad).Membranes were blocked with blocking buffer containing 2% BSA or 5%fat-free dry milk in Tris-buffered saline solution and Tween 20 (10 mMTris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween 20) and incubated overnight at4° C. with primary antibody diluted in blocking buffer. Incubations withHRP conjugated secondary antibodies (1:5000) were performed for 1 h atroom temperature and visualization was performed using chemiluminescence(ECL).

Image Acquisition and Analysis—

All images were acquired on an inverted Zeiss LSM510 confocal microscopewith LSM acquisition software (Carl Zeiss Microscopy). Forquantification of P-PKCζ T410 immunoreactivity in cell cultures,exposure time was adjusted to be under the saturation level for thehighest immunofluorescence signal and was conserved between thedifferent samples. Mean immunofluorescence intensity level in neuronalcell bodies was measured using FiJi-ImageJ software (NIH). Allquantifications were performed on 10 non-overlapping fields in cellcultures from at least three independent experiments and on fournon-consecutive sections from at least three animals for in vivoexperiments.

Statistical Analysis—

Statistical analyses were performed using ANOVA with Bonferroni Posttestor Student's t test using GraphPad Instat 3.05 (GraphPad Software). Alldata are expressed as means or percents±s.e.m. *p<0.05, **p<0.01,***p<0.001.

Example 2 Inhibition of aPKC Promotes Axonal Degeneration and NeuronalApoptosis

A culture system was established to investigate the expression and roleof aPKC in the survival of cerebral cortical neurons. E16.5 corticalcells were cultured for 3 days in vitro (DIV3) and double immunolabeledfor the pan-axonal marker SMI-312 and the dendritic marker MAP2. At thisstage, most neurons exhibit a long process labeled for SMI-312corresponding to the axon and few short MAP2+ processes that will giverise to the dendrites. To evaluate the proportion of polarized neuronsat this stage, the number of neurons with a long process labeled forSMI-312 and not labeled for MAP2 in its distal part was counted over thetotal number of SMI-312+ neurons. It was found that 84.2±3.45% ofSMI-312+ neurons were polarized in these culture conditions. Then, aPKCexpression was investigated in cortical neurons by using an antibodythat recognized all isoforms of aPKC family including PKCζ, PKCλ/ι andPKMζ (34). These observations indicate that aPKC immunolabeling waslocalized in neuronal cell bodies and SMI-312+ axons (FIG. 1A).

To investigate the role of aPKC in neuronal survival, aPKC activity wasinhibited by transfecting a kinase-defective PKCζ (PKCζ-KD) or anonphosphorylatable mutant (PKCζ-T410A) in cortical neuron cultures.aPKC inhibition by these constructs induced neurite degeneration (FIGS.1B, 1C, and 1E) after 2 days of transfection, as shown by the massivefragmented neurites of PKCζ-KD and PKCζ-T410A transfected neurons (FIGS.1B and 1C). PKCζ-KD and PKCζ-T410A overexpression also induced neuronalapoptosis as shown by the increase of the proportion of aCasp3+ neurons(FIGS. 2D and 2F). Overexpression of PKCζ-WT protein did not affectneurite integrity or neuronal survival (FIGS. 1B-1F). The massive axonfragmentation suggests that axons have grown extensively initially andunderwent degeneration all at once, suggesting that aPKC is required foraxon maintenance. Interestingly, while overexpressed PKCζ was localizedin both neuronal cell bodies and neurites, overexpressed PKCζ-KD andPKCζ-T410A mutant proteins were excluded from the processes and wererestricted to the cell bodies (FIG. 1B), suggesting that PKCζ activitymight regulate its own localization and/or the stability of the protein.

In order to test the role of aPKC in axon survival using an independentmethod and that allows biochemical analyses, a cell-permeablemyristoylated pseudosubstrate specific to aPKC was used (14). As acontrol, a myristoylated pseudosubstrate specific to conventional PKC(14) was used. The IC₅₀ of aPKC and cPKC inhibitors are 10 μM and 8 μMrespectively, so a starting dose of 10 μM was used. At this dose, aPKCinhibitor but not cPKC inhibitor promotes rapid axonal degeneration incortical neurons as shown by the beading aspect of SMI-312+ axons, 2 hafter aPKC pseudosusbtrate treatment (FIGS. 2A and 2B). Furthermore,aPKC pseudosubstrate promotes axonal degeneration in a dose- (FIG. 2C)and time-dependent manner (FIG. 2D), with an effect starting at 5 μM andas early as 1 h at a dose of 10 μM. To verify the efficacy of aPKCpseudosubstrate, P-PKCζ-T410 immunoreactivity was measured in neuronalcell bodies after 2 h of 10 μM aPKC PS treatment and it was found thatit was significantly decreased (FIG. 2E), indicating that aPKCpseudosubstrate decreases (auto)-phosphorylation activity of aPKC.

Axon degeneration induced by aPKC pseudosubstrate occurred as early as 1h (FIG. 2D) and the time course analysis revealed that almost allneurons exhibited degenerating axon or were devoid of axon by 4 h (FIGS.2F and 2G). In parallel to axon degeneration, neuronal cell body deathwas measured using TUNEL and activated Caspase-3 staining at 2 h, 4 hand 24 h. It was found that cell death in SMI312+ neurons started at 4 hof incubation with aPKC pseudosubstrate (FIG. 2H), when thequasi-totality of axons had already degenerated (FIG. 2G), suggestingthat blocking aPKC signaling triggers a dying back mechanism.Interestingly, neurons started to show aCasp3 after 4 h but all neuronsdid not express activated Caspase-3 by 24 h (FIG. 2I) while all of themwere TUNEL+ by 24 h (FIG. 2H), suggesting that aPKC inhibition maytrigger both caspase-3-dependent and -independent cell death pathways.

Example 3 aPKC Inhibition Destabilizes Microtubules by Regulating MARK2Activity and Tau Phosphorylation

Microtubule degradation is an early cellular event in axon degenerationprocess, preceding axonal beading and neurofilament fragmentation (1,35-37). It was hypothesized that inhibition of aPKC may promote rapidaxon degeneration by destabilizing microtubules. To test thishypothesis, cortical neuronal cell cultures were first pre-incubatedwith taxol, a microtubule stabilizer (38), 2 h prior to and during aPKCpseudosubstrate treatment. Axon degeneration induced by aPKC inhibitionwas reduced when neuronal cell cultures were pre-treated with taxol(FIGS. 3A and 3B). Using specific markers for the different compartmentsof the neuronal cytoskeleton (Tau for microtubules, SMI-312 forneurofilaments and rhodamine-phalloidine for F-actin filaments), it wasfound that microtubules and neurofilaments, but not actin filaments,were disrupted in axons of neurons treated with aPKC pseudosubstrateusing confocal microscopy. These observations suggest that aPKC isrequired for microtubule stability and that aPKC inhibition promotesboth microtubule and neurofilament disruption, ultimately leading toaxonal degeneration.

The signaling pathway involved in microtubule destabilization induced byaPKC inhibition was further investigated. First, the efficacy of aPKCpseudosubstrate on blocking aPKC (auto-)phosphorylation activity wasconfirmed by western blotting. aPKC pseudosubstrate decreased P-PKCζT410 level after 2 h of incubation (FIG. 3C), in agreement with theimmunocytochemical analyses (FIG. 2E). Then the focus was on MARK2because (1) it belongs to the MARK/Parl family of kinases that wereoriginally discovered to trigger microtubule disassembly (39) and (2) itis inhibited when phosphorylated by aPKC on T595 (40). It was found thataPKC pseudosubstrate treatment reduced phosphorylation of MARK2 on T595at 2 h (FIG. 3D), indicating that MARK2 activity was increased when aPKCwas inhibited. Furthermore, Tau phosphorylation in the microtubulebinding domain at S262 (FIG. 3E) was increased at 2 h while stablemicrotubules, revealed by Glu-Tubulin (FIG. 3F), were decreased. Theseobservations are in agreement with previous studies showing that MARKfamily kinases destabilize microtubules by phosphorylating MAPsincluding Tau (9, 39, 41).

To further test the role of Tau phosphorylation in axonal degenerationinduced by aPKC inhibition, cortical neurons were co-transfected withPKCζ-KD and Tau-WT or a non phosphorylatable mutant Tau at S262(Tau-S262A) plasmidic construct. Overexpression of Tau-WT, but notTau-S262A, induced axonal degeneration and neuronal apoptosis in asimilar extent than PKCζ-KD (FIGS. 3G-3I). Co-transfection of PKCζ-KDwith Tau-S262A but not with Tau-WT prevented axonal degeneration andneuronal cell death induced by PKCζ-KD overexpression (FIGS. 3G-3I),suggesting that axonal degeneration induced by PKCζ-KD is mediated byTau phosphorylation at S262.

Example 4 aPKC Inhibition Activates the JNK-cJun Signaling Pathway

JNKs/SAPKs are a subfamily of MAPKs involved in a variety ofphysiological and pathological processes in the central and peripheralnervous system. In particular, JNKs mediate neuronal cell death inresponse to stress and injury and in some pathological conditions (42,43). JNKs were also shown to induce, retrograde axonal degeneration andto limit motor recovery after spinal cord injury (44). It was thereforesought to determine whether JNKs might be involved in axonaldegeneration induced by aPKC inhibition. Western-blotting experimentsrevealed that aPKC inhibition induced by aPKC PS leads to an increase ofphosphorylation of JNKs at T183/T185. Increased phosphorylation occurredon both p54 and p46 protein isoforms at 1 h (FIGS. 4A and 4B).Phosphorylation of the transcription factor c-Jun was increased at S63,at 1 h and 2 h (FIGS. 4A and 4C), indicating that c-Jun, a directsubstrate of JNK (45), is quickly activated when aPKC activity isinhibited. Increase of P-JNK-T183/T185 and P-cJun-S63 was confirmed inthe nucleus of neurons by immunocytochemistry (FIGS. 4D and 4E). Theseresults suggest that aPKC inhibition might promote axonal degenerationand neuronal cell death by activating the cell death JNK/c-Jun pathway.

Example 5 Ryk Promotes Axonal Degeneration Induced by aPKC Inhibition

Previous studies showed that aPKC is a kinase required for Wnt-Frizzled3mediated axon growth and attraction and also exerts a positive feedbackfunction to amplify planar cell polarity signaling by increasingFrizzled3 endocytosis (46). Because Ryk is a repulsive Wnt receptor andinhibits planar cell polarity signaling, it was tested whether Rykregulates aPKC. It was found that P-aPKC-T410 level measured byWestern-Blotting was increased in Ryk KO cortical neurons cultured for 3days (FIGS. 5A and 5B), suggesting that Ryk normally inhibit aPKCactivity.

It was previously shown that expressions of certain Wnts and of the Rykreceptor are re-induced after spinal cord injury and that blockingWnt/Ryk signaling reduces the retraction of corticospinal axons from theinjury site (32). These recent studies indicate that Ryk expression isincreased in motor neurons and axons in the spinal cord of a mouse modelof ALS at early stage of the disease progression, suggesting that Ryk isinvolved in the early steps of neurodegeneration in ALS (47). It wastherefore hypothesized that Ryk may be also involved in axondegeneration and it was tested whether blocking Wnt/Ryk signaling couldprotect cortical axons from degeneration induced by aPKC inhibition.

To block Ryk signaling, a mouse monoclonal antibody against theectodomain of mouse Ryk was generated (by the laboratory of AlexKolodkin at John Hopkins University, Baltimore, Md.). To test thespecificity of this antibody, Western-Blotting analyses were performedon protein extracts from HEK cells transfected with a mouse Rykexpression vector. A signal was detected at approximately 70 KDa, theexpected size for Ryk protein. A weaker band of similar size wasdetected endogenously in E14.5 embryonic wild type mouse tissue but notin Ryk KO tissue, suggesting that the antibody is specific to Ryk.Pre-incubation of cortical neuronal cell cultures with the mouse Rykantibody but not with normal mouse IgG, 2 h prior to and during the 2 hof aPKC PS treatment, partially blocked axonal degeneration (FIGS. 5Cand 5D). Axonal degeneration induced by aPKC inhibition was alsoanalyzed in Ryk KO mouse neuronal cell cultures. Axonal degenerationtriggered by aPKC PS was reduced in Ryk KO cortical neurons compared toWT neurons (FIGS. 5E and 5F).

Example 6 Neuronal Cell Death is Reduced in the Retrosplenial Cortex ofDeveloping Ryk KO Mice

The in vitro results suggest that Ryk may be involved in neuronal celldeath. Previous studies showed that Ryk is expressed in the developingrodent forebrain including the isocortex, hippocampus and striatum (24,48, 49). Apoptosis was examined in several regions of the forebrain inE18.5 Ryk deficient embryos, using aCasp3 immunostaining. Ryk KO miceexhibit craniofacial abnormalities including cleft palate and die atbirth (50), so all analyses were performed prenatally during lategestation on E18.5 embryos. At E18.5, the overall architecture ofRyk^(−/−) brain appears roughly normal and undistinguishable fromRyk^(−/−) and Ryk^(+/−) embryos (27). The number of aCasp3+ cells,although low in WT embryos was decreased by 45% in E18.5 RykKO mice in arestricted area of the cortex, the RSP, which corresponds to theposterior part of the cingulate cortex (FIGS. 6A-6C). However, apoptosiswas not changed in the dorsolateral cortex, hippocampus and striatum inRyk KO embyos, suggesting that Ryk might regulate cell death in specificregions of the developing cortex.

Example 7 Ryk Heterozygosity Attenuates Neuronal Cell Death in theRetrosplenial Cortex of Frizzled3 KO Embryos

Previous studies showed extensive cell death in the striatum of mouseembryos deficient for the Wnt receptor Frizzled3 and that Frizzled3 wasstrongly expressed in the developing striatum and isocortex (51). Celldeath was examined in Frizzled3 KO mice, in the striatum and otherregions of the forebrain including the RSP. These observations confirmedthat apoptosis was strongly increased in the striatum. Interestingly, itwas found that apoptosis was increased by 5 fold in the RSP of E18.5Frizzled3^(−/−) embryos, compared to Frizzled3^(+/+) and Frizzled3^(+/−)mice (FIGS. 6D and 6E). Double immunolabelings with neural precursormarker nestin and early neuronal marker βIII-tubulin showed that aCasp3+immunolabeling was localized in βIII-tubulin+ neurons. More precisely,aCasp3 co-localized with CTIP2 which is expressed in neurons of corticallayer V during embryonic development. However, apoptosis was notsignificantly different in E18.5 Frizzled3^(−/−) compared toFrizzled3^(+/+) embryos in the dorsolateral part of the cortex andhippocampus.

Since cell death was regulated in an opposite manner in the RSP ofFrizzled3 KO and Ryk KO embryos, Frizzled3^(+/−) mice were crossed withRyk^(+/−) mice. Double heterozygous mice resulting from these crossingswere crossed with Frizzled3^(+/−) mice for 6 generations to get theFrizzled3/Ryk mouse line into the Frizzled3 strain (Black6). Then,Frizzled3^(+/−) Ryk^(+/−) females were crossed with Frizzled3^(+/−)Ryk^(+/−) males. Over 52 embryos from 7 litters did not produce anyembryos double knock-out for Frizzled3 and Ryk at E18.5, suggesting thatFrizzled3^(−/−) Ryk^(−/−) embryos might not be viable at this developingstage. The analyses were therefore performed in Frizzled3^(−/−)Ryk^(+/−) embryos and it was found that knocking down Ryk expression by50% attenuated apoptosis in the RSP of Frizzled3 KO embryos at E18.5, asshown by a 30% reduction of the number of activated Caspase 3+ cells inthe RSP of Frizzled3^(−/−) Ryk^(+/−) embryos compared to Frizzled3^(−/−)Ryk^(+/+) animals (FIGS. 6D and 6E).

Example 8 Additional Methods

Animals were housed on a 12 hr light/dark cycle and behavioral analyseswere done at consistent morning hours during the light cycle. Both miceand rats were group housed, except for mice with cranial windows whichwere singly housed after window implantation. Group sample sizes werechosen based upon previous studies and power analysis (25% effect size,a=0.05, 639˜0.2, power of 80%). One mouse was excluded from the studydue to evidence of incomplete CS lesion with labeled corticospinal axonspresent at and below the level of the injury and one mouse was excludedas it did not attempt to perform the behavioral task after injury. Allprocedures and methods (surgical procedures, behavioral assays, tissueprocessing, immunostaining, image analysis, COS-7 transfection, andcortical mapping) were performed by investigators blinded to genotype ortreatment group.

Generation of Transgenic Mice—

The target vector containing loxP-flanked exons 3-6 as well as thePKG-neo selection cassette was transfected into ES cells. Cells werescreened by Southern blot and PCR for integration of the targetingvector. Chimeric mice were then generated. Ryk cKO mice were crossedwith Ai14 B6.Cg mice containing a loxP-flanked stop cassette preventingtdTomato expression (The Jackson Laboratory, Bar Harbor, Me.,RRID:IMSR_JAX:007914) and backcrossed into C57BL/6J for 6 generations.For cortical mapping experiments, Ryk cKO::tdTomato Ai14 mice werecrossed with mice expressing channelrhodopsin (Chr2) behind the Thy1promoter (B6.Cg-Tg(Thy1-COP4/EYFP)18Gfng/J) (The Jackson Laboratory).

Surgical Procedures—

Cortical AAV injection: Adult female C57BL/6J mice (6.1±0.1 weeks old)were deeply anaesthetized with isoflurane until unresponsive to toe andtail pinch and the area over the skull was shaved and cleaned withpovidone-iodine before incision. The skull was thinned bilaterally overthe motor cortex and self-complementary AAV2/6 Cre-HA (Salk Institutefor Biological Studies Gene Transfer, Targeting and Therapeutics Core,La Jolla, Calif.) (1.49×10¹¹ genome copies/ml) was injected into 10sites per hemisphere (250 nl/site) with a 36 ga NanoFil needle (WorldPrecision Instruments Inc., Sarasota, Fla.).

Cranial Window:

Adult female C57BL/6J mice (7.4±0.2 weeks old) were deeply anaesthetizedwith isoflurane. The skin over the skull was removed, the skullsurrounding the motor cortex contralateral to the dominant forelimb wasthinned, the skull over the motor cortex was removed, andself-complementary AAV2/6 Cre-HA was injected to 10 sites, as above. Theexposed cortex was covered with a 5 mm #1 round glass coverslip (WarnerInstruments, Hamden, Conn.) secured with VetBond (3M, St. Paul, Minn.).The exposed skull was covered with dental grip cement (Dentsply, York,Pa.).

CS and C3 Dorsal Column Lesion (Mice):

Mice were deeply anaesthetized with isoflurane, spinal level CS (or C3)was exposed by laminectomy and the dorsal columns were lesioned at adepth of 1 mm with Vannas spring scissors (Fine Science Tools, FosterCity, Calif.). The dorsal musculature was sutured with 4-0 silk suturesand the skin was closed with wound clips. Mice were randomly selectedfor C3 lesion or C3 sham (laminectomy only) groups.

Pyramidotomy:

Mice were deeply anesthetized with ketamine (120 mg/kg) and xylazine (12mg/kg), an incision was made and the ventral musculature was pushedaside to expose the pyramids. The dura was opened and the pyramidipsilateral to the craniotomy was lesioned by 15° microscalpel (ElectronMicroscopy Sciences, Hatfield, Pa.) as previously described.

CS Dorsal Column Lesion (Rats):

Adult female Fischer 344 rats (120-135 g) were deeply anaesthetized with2 ml/kg of ketamine cocktail (25 mg per ml ketamine, 1.3 mg per mlxylazine and 0.25 mg per ml acepromazine). Spinal level CS was exposedby laminectomy, the dura was punctured over the dorsal horn, and thedorsal columns were lesioned with 2 passes of a Scouten wire-knife(David Kopf Instruments, Tujunga, Calif.). The dorsal musculature wassutured with 4-0 silk sutures and the skin was closed with wound clips.Polyethylene intrathecal catheters (Durect Corp., Cupertino, Calif.)were pre-filled with either mouse IgG or Ryk monoclonal IgG (clone25.5.5 generated against the ectodomain of Ryk, amino acid range 90-183,by Johns Hopkins Monoclonal Antibody Core, Baltimore, Md.) (1 mg/ml) inartificial cerebrospinal fluid, threaded through magna cisterna to thecervical spinal cord, secured with 4-0 silk sutures, and attached tomodel 2004 osmotic minipumps (Durect Corp.) filled with 2000 mouse IgGor Ryk IgG. Rats were randomly selected for mouse IgG or Ryk monoclonalIgG treatment. Osmotic minipumps were removed after 28 days. At 16 weeksafter CS spinal cord injury, rats were injected bilaterally with 10%wt/vol 10,000 MW biotinylated dextran amine (BDA) in sterile phosphatebuffered saline at 20 sites per hemisphere (250 nl/site); animals weresacrificed 2 weeks later.

Cortical Mapping:

Mice were lightly anaesthetized with ketamine (100 mg/kg) xylazine (10mg/kg) mixture, still responsive to toe and tail pinch, and maintainedduring the course of stimulation with ketamine/xylazine mixture. Micewere fixed in a stereotaxic frame (David Kopf Instruments) and a fiberoptic cable and cannulae (200 μm diameter) affixed to the stereotax armwere used to stimulate motor cortex locations relative to bregma thatwere unobstructed by skull or dental cement (maximum of 165 sites).Stimulation was 3 pulses of 470 nm light, 250 ms duration at 1 Hz from asingle channel LED driver (Thorlabs, Newton, N.J.). The intensity ofstimulation was increased from 50 mA up to 1000 mA until movement wasdetected in 3 consecutive pulses. Sites with no evoked movements at 1000mA were scored as unresponsive. Only contralateral forelimb movementswere scored; occasional, weaker, ipsilateral movements were observed aspreviously described in rats.

Ryk Knockout in Postnatal Day 0 Pups:

Pups were injected with 0.50 AAV2/1-synapsin-Cre (Penn Vector Core,Philadelphia, Pa.) (1.99×10¹³ genome copies/ml) was injected into 2sites in the motor cortex unilaterally with a 36 ga NanoFil needle(World Precision Instruments Inc.). Mice were sacrificed 7 days laterand the motor cortex was isolated and homogenized in lysis buffer (20 mMTris HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 10 mMβ-glycerophosphate, 1 mM Na₃VO₄, 0.5% wt/vol sodium dodecyl sulfate, 1%vol/vol TritonX-100, and complete protease inhibitor cocktail (Roche,Indianapolis, Ind.). Protein was analyzed by Western blot (40 μg/well).Antibodies used for Western mouse anti-Ryk (20 μg/ml) (Johns HopkinsMonoclonal Antibody Core), GAP DH (1:1000) (EMO Millipore, Billerica,Mass., catalog #MAB374, RRID:AB_2107445).

Sacrifice and Tissue Processing:

Animals were deeply anaesthetized with ketamine cocktail, transcardiallyperfused with ice-cold PBS followed by 4% wt/vol paraformaldehyde inPBS, and brains and spinal columns were post-fixed overnight at 4° C. in4% wt/vol paraformaldehyde. Tissue was cryoprotected in 30% wt/volsucrose in PBS. Mouse spinal cords and brainstems, and rat brainstemswere sectioned on a cryostat (Leica, Buffalo Grove, Ill.) at 20 μm(saggital spinal cords, transverse brainstems) and mounted directly onSuperfrost Plus slides (Fisher Scientific, Pittsburgh, Pa.). Rat spinalcords were sectioned sagittally at 40 μm thick and collected asfree-floating sections. Sections were washed three times with PBS,blocked for one hour in PBS with 0.25% triton-X100 (PBST) and 5% donkeyserum, then incubated overnight at 4° C. with primary antibodies in PBSTplus 5% donkey serum. The next day, sections were washed three times,incubated with Alexa Fluor conjugated secondary antibodies (LifeTechnologies, Grand Island, N.Y.; Jackson ImmunoResearch, West Grove,Pa.) for 2.5 hours at room temperature, counterstained with DAPI (1μg/ml) (Sigma-Aldrich, St. Louis, Mo.) and washed three final times inPBS. Antibodies used for fluorescent immunohistochemistry were: rabbitanti-dsRed (1:1000) (Clontech Laboratories Inc., Mountain View, Calif.,catalog #632496, RRID:AB_10013483), monoclonal G-A-5 anti-GFAP (1:200)(Sigma-Aldrich, catalog #G3893, RRID:AB_2314539), guinea pig anti-vGlut1(1:1000) (EMO Millipore, catalog #AB5905, RRID:AB_2301751), and rabbitanti-GFAP (1:750) (Dako, Carpinteria, Calif., catalog #Z0334,RRID:AB_10013382).

COS-7 Cell Transfection:

COS-7 cells were transfected with pcDNA4-Ryk using FuGene6 (Roche) toexpress full length Ryk. Cells were either fixed for 30 min withice-cold 4% wt/vol paraformaldehyde in PBS for immunocytochemistry, orlysed with lysis buffer for Western blot.

Image Acquisition and Analysis:

Images were acquired on an inverted Zeiss LSM510 confocal microscopewith LSM acquisition software (Carl Zeiss Microscopy, LLC, Thornwood,N.Y.). Image density quantification was done on thresholded images usingImageJ (NIH, Bethesda, Md.). An investigator blinded to the experimentalgroup performed all analyses. Axon index is the total thresholded pixelsat every 0.411 μm in 8 total serial sagittal spinal cord cryosectionsspaced 140 μm apart for mice, or 0.741 μm in 6 total serial sagittalspinal cord cryosections spaced 280 μm apart for rats, divided bythresholded pixels in transverse sections of the pyramid at the level ofthe obex. Lesion volume was calculated using the Cavalieri estimatortool in StereoInvestigator (MBF Bioscience, Williston, Vt.) on everyseventh 40 μm sagittal section. For tdTomato and vGlut1 colocalization,all axons within the gray matter were quantified over a region 210 μmwide at a distance of 600 μm rostral to the CS lesion in the 8 totalserial sagittal cryosections used for tdTomato quantification. Thelocation of 600 μm was chosen as it was observed that the highestdensity of axon collaterals in this region in both groups of animals(FIG. 8G) and it was located between the original CS and secondary C3lesions.

Behavioral Testing:

All animals were trained on skilled forelimb reach over a period of twoweeks prior to bilateral spinal cord injury. Animals were foodrestricted during training and then for 24 hours prior to weeklytraining after injury. Animals reached through a vertical slot in thefront of an acrylic chamber and over a small gap to retrieve a rewardpellet. Mice performed 25 reaches per session for 20 mg sucrose rewardtablets (TestDiet, St. Louis, Mo.). Rats performed 50 reaches persession for 45 mg sugar pellets (Bio-Serv, Flemington, N.J.). Successfulretrieval rate was calculated as the number of pellets that wereretrieved and eaten divided by the number contacted by the forepaw.Forelimb reach was trained twice weekly by two independent investigatorsblind to genotype or experimental treatment; the two independent scoreswere averaged. Mice in the group without weekly training during recoverywere only tested twice, at 8 weeks after injury. In addition to forelimbreach, rats were tested once weekly on a grid crossing task, whereforelimb footfalls were calculated as a percentage of total forelimbsteps in 3 passages over a 60 inch span of 1 inch equidistant wire grid.

Statistics:

Statistical tests indicated in main text were performed using JMP 9software (SAS Institute, Cary, N.C.). It was previously demonstratedthat inhibition of repulsive Wnt signaling results in sprouting andplasticity of descending corticospinal and ascending dorsal columnsensory axons after spinal cord injury. In addressing the hypothesisthat Ryk cKO or Ryk monoclonal antibody enhanced axon sprouting, theincreases were tested by one-tailed t-test (FIGS. 8E, 10B, 12I, and 18).In order to test longitudinal behavioral studies with multiple, equallyspaced measurements, repeated measures ANOVA were utilized (FIGS. 8F,12B, and 21). In testing multiple groups with continuous, parametricdata, ANOVA with post-hoc Bonferroni correction was utilized onappropriate post-hoc comparisons (FIGS. 11C and 15C). Bivariatecorrelation was performed to determine the relationship between forelimbfunction and cortical maps (FIG. 15D). Continuous data was tested withparametric tests and data was assumed to be normally distributed, butthis was not formally tested.

Example 9 Ryk cKO Enhances Recovery of Fine Motor Control after SCI

Mice underwent two weeks of training for the reaching and grasping task,followed by a CS dorsal column spinal cord lesion: a partial spinal cordinjury model leaving the dorsal gray matter, lateral white matter andthe entire ventral spinal cord intact (FIGS. 8A and 8D). Immediatelyafter dorsal column lesion, forelimb reaching and grasping function islost (FIG. 8F). With continued training, the success rate of sugarpellet retrieval recovers due to reconfiguration of neural circuits. Ithas been shown that the CST undergoes robust collateral sprouting afterinjury and some of the new sprouts are thought be responsible for newfunctional circuits. However, axon sprouting is inhibited by molecularcues that limit axon plasticity.

Members of the Wnt glycoprotein family are phylogenetically conservedaxon guidance molecules that direct the growth along the rostro-caudalaxis of both ascending sensory axons and descending CST axons duringdevelopment. The repulsive Wnt receptor, Ryk, which mediates Wntrepulsion of the developing CST neurons is either not expressed innormal adult motor cortex and the CST neurons or expressed at extremelylow levels to be detected by in situ hybridization orimmunobistochemistry. Spinal cord injury re-induces expression of RykmRNA and protein in the injured CST. By injecting function-blockingantibodies to Ryk and diffusible Wnt inhibitors, it was found thatinhibiting Wnt-Ryk signaling enhanced the plasticity of both sensory andmotor axons following injury. However, Ryk antibodies or Wnt inhibitorsmay exert the effects by impacting on the environment, such as the glialcells around the lesion, rather than CST axons per se.

To specifically test the role of Ryk in neurons, a Ryk conditionalallele (cKO) was created and these mice were crossed with Ai14 86.Cgmice containing a loxP-flanked stop cassette preventing tdTomatoexpression, in order to specifically label recombined corticospinalaxons after viral transduction (FIGS. 8B and 8C). Ryk cKO::tdTomato Ai14mice were injected with an adeno-associated virus (AAV) that expressesCre recombinase under the control of the cytomegalovirus (CMV) into theprimary motor cortex and the enhancement of corticospinal circuitremodeling was assessed (FIG. 8A). AAV-Cre was injected to adult motorcortex an average of 2.3 weeks prior to CS dorsal column lesion in orderto ensure sufficient time for Cre expression, so that injury would notlead to Ryk expression. It was found that Ryk deletion in the CSTsignificantly enhanced recovery of skilled forelimb function as assessedby forelimb reach over a period of 12 weeks (Repeated measures ANOVAP<0.005, FIGS. 8F and 16). The effects of Ryk deletion were observedearly on, with a trend towards better performance at early testingsessions. This was consistently observed and may be due to reducedretraction of axons and collaterals as previously demonstrated at 5weeks post-injury in animals infused with Ryk antibodies. Following Rykconditional deletion, mice recovered to 81±7% of peak pre-injury successrates, compared to only 60±5% in control mice.

Example 10 Ryk cKO Enhances CST Collateral Sprouting after SCI

To begin to address the mechanisms underlying improved functionalrecovery in Ryk cKO mice, CST collaterals and synapse density wereanalyzed along collateral sprouts in the cervical spinal cord. It wasfound that conditional Ryk deletion did not significantly reduce axonaldie-back of the injured CST (one-tailed t-test P=0.12), but did lead tosignificantly increased numbers of CST collaterals within the spinalgray matter, both rostral and caudal to the site of CS injury, at 12weeks post-injury (one-tailed t-test P<0.05, FIG. 9E). In addition to agreater number of axon collaterals, mice with Ryk conditionally deletedfrom cortical pyramidal neurons exhibited pre-synaptic vesicularglutamate transporter 1 (vGlut1)-labeled puncta on identifiedcorticospinal axon collaterals at 600 μm rostral to the injury site,suggesting enhanced functional connectivity following Ryk conditionaldeletion (FIGS. 10C and 10D).

The majority of CST axons reside within the dorsal columns and arelesioned by the CS dorsal column injury. A sparse, minor, component ofCST axons descend down the spinal cord within the lateral columns(lateral CST), which remained intact in the CS lesion paradigm and maycontribute to functional recovery (FIG. 8D). To test this, thedistribution of axon collaterals were first characterized in the spinalcord both rostral and caudal to the CS lesion. It was observed that anincrease in axon collateral density in Ryk conditional deleted micethroughout the gray matter, with the highest density more medial, inclose proximity to the principal dorsal column corticospinal tract(FIGS. 10A and 10B), suggesting that increased branches may come fromthe dorsal column CST following Ryk deletion. No axons were presentwithin the dorsal column CST caudal to CS lesion in any of the micestudied.

To further assess the contribution of the dorsal column CST tobehavioral recovery, a second dorsal column lesion was performed inthese animals 12 weeks after CS injury at the C3 level, 1.15±0.07 mmrostral to the original CS injury (FIGS. 11A and 11B). The lateral CSTis again spared in this lesion. After a one-week delay to allow the miceto recover from the immediate hyporeflexic stage of spinal shock,behavioral testing began. The secondary injury at C3 ablated theenhanced functional recovery that was observed in the Ryk conditionaldeletion mice, leaving only the modest levels of partial recoveryachieved by control mice (FIG. 11C). This suggests that the axon sproutsfrom the dorsal column CST are indeed responsible for the enhancedfunctional recovery in the Ryk conditional knockout. This also suggeststhat the spared lateral CST axons may provide only a minor contributionto a basal level functional recovery independent of the dorsal columncorticospinal tract. Quantification of axon distribution at 2 weeksafter C3 lesion confirmed that the secondary C3 injury eliminated amajority of axon collaterals between the two injury sites, therebydisrupting the remodeled corticospinal circuit (FIGS. 11D-11F).

Example 11 Monoclonal Ryk Antibody Promotes Functional Recovery

To test whether inhibition of the Wnt-Ryk signaling axis in the injuredspinal cord after spinal cord injury is sufficient to increase CSTremodeling and enhance behavioral recovery, a new monoclonal Rykantibody was generated using half of the Wnt binding domain (amino acidrange 90-183) as the antigen and infused into adult rats immediatelyfollowing spinal cord injury (FIGS. 12A, 17A and 17B). Function-blockingpolyclonal antibodies were previously generated using the same region.Following a CS dorsal column wire-knife lesion, Ryk antibody infusionvia osmotic minipump for 4 weeks promoted recovery of skilled forelimbfunction in the forelimb reach task with all rats recovering to peakpre-injury levels, as compared to only half of rats infused with IgGcontrol (FIG. 12B). Ryk antibody did not enhance recovery in the gridcrossing locomotor task since rats exhibited similar levels of forelimbstepping impairment irrespective of treatment group (FIG. 12C).Corticospinal axons were labeled with biotinylated dextran amine (BDA)injections into motor cortex. Consistent with the conditional Rykdeletion prior to injury, it was found that the Ryk monoclonal antibodyinfusion at the time of injury resulted in an increase in corticospinalaxon collaterals both rostral and caudal to the level of injury(one-tailed t-test P<0.05, FIGS. 12E-12I and 18). The extent of increaseof collateral sprouts after Ryk antibody infusion in rats was similar tothat observed in CST axons lacking Ryk expression in mice (FIGS. 9E and12I). These results also suggest that Ryk signaling is a feasibletherapeutic target, since functional recovery can be promoted byblocking its function after spinal cord injury.

Example 12 Cortical Map Re-Organization During Recovery

In order to address the circuit mechanisms with which the primary motorcortex regains control over the remodeled spinal cord, an optogeneticsapproach was used to monitor cortical output. Cortical motor maps havebeen studied using intracortical electrical stimulation in rodents andprimates, as well as transcranial magnetic stimulation in humans. Recentadvances in optogenetic tools allow for the stimulation of specificneural populations in a minimally invasive manner. Specifically, theexpression of the light-activated, non-selective, cation channelchannelrhodopsin-2 (ChR2) under control of the Thy1 promoter (Thy1-ChR2)allows for selective activation of layer V projection neurons within themotor cortex. Unilateral craniotomies were performed on Thy1-ChR2 micecontralateral to the dominant forelimb in order to investigate motor mapchanges through repeated optogenetic mapping of evoked motor outputafter injury (FIG. 19). Following craniotomy, AAV-Cre was injectedunilaterally into the motor cortex contralateral to the dominantforelimb as contralateral cortex exhibits motor plasticity in responseto forelimb training; and CST collateral plasticity, which supportsfunctional recovery, was specifically induced in CST axons with Rykdeletion. Motor map output was assessed by observing evoked,contralateral motor outputs in sedated mice.

Massive remapping of cortical motor output was observed immediatelyafter spinal cord injury in the mouse. Acutely (3 days) after CS injury,the total area of motor representations for limb muscles at or below thelevel of lesion was reduced or eliminated (FIG. 12C). Conversely, motormaps expanded for muscle groups with motor neurons above the injurysite, most notably elbow flexion mediated by biceps brachii andbrachialis (FIGS. 13B, 14E, and 20C). Over the next two months followingspinal cord lesion, cortical maps underwent gradual changes withcontinued forelimb reaching and grasping training (FIG. 20). Behavioralrecovery after CS spinal cord injury plateaued between 4 and 8 weekspost-injury (FIGS. 8F and 21), with a median time to reach 90% of peak,post-injury, performance of either 6 weeks in control mice or 5 weeks inRyk conditional deletion. Therefore, cortical motor maps were examinedbefore and after peak recovery at 4 weeks and 8 weeks, respectively,post-injury. At 4 weeks post-injury, significant differences wereobserved in the proportion of motor cortex allocated to forelimbextensor (biceps) or forelimb flexor (triceps) activation, with Rykdeleted mice exhibiting larger flexor motor maps at the expense ofreduced extensor maps (P<0.05 one-tailed t-test, FIGS. 14E and 14F).Expansion of elbow extensor areas at 4 weeks into regions originallyoccupied by the flexor was inversely correlated with behavioral recovery(n=21 mice: 10 (control), 11 (Ryk cKO), Spearman's p=−0.5766, P=0.0062).By 8 weeks post-injury, Ryk deleted mice exhibited a similar pattern ofextensor and flexor motor maps as controls, however the total areaoccupied by all elbow movements (flexor and extensor) was significantlylarger in Ryk deleted mice (one-tailed t-test, P=0.0480 04)=1.79).Additionally, at 8 weeks post-injury, wrist flexor representationsreturned to (or exceeded) maximal pre-injury size in 64% of Ryk deletedmice compared to 10% of control mice (Wilcoxon rank sum P=0.0136χ²=6.086, FIG. 20C). Recovery of wrist flexor control correlated withimprovement of forelimb reach performance at 8 weeks post-injury(Spearman's p=0.4555, P=0.0380). Over the course of the experiment,there was a strong correlation of wrist movement and skilled forelimbreach performance, regardless of injury or genotype (Pearson's p=0.665,P<0.0001, FIG. 15D).

In order to further characterize the remapped cortical output, mice weresubjected to a second dorsal column lesion at C3, rostral to the levelof extensor motor units, at 8 weeks after CS injury (FIG. 11B). It wasfound that the C3 injury significantly reduced flexor motor maps in allmice but, surprisingly, had little effect on the recovered extensormotor maps, suggesting the flexor control is routed from connectionsrostral to C3 or from the lateral corticospinal tract (FIG. 13D).Importantly, a subsequent unilateral pyramidotomy abolished unilateralforelimb responses to cortical stimulation and also the ability of miceto perform the forelimb reach task (FIGS. 13D and 21). Althoughplasticity of other supraspinal pathways, such as the rubrospinal tractor reticulospinal tract may also contribute to functional recovery, theeffects of unilateral pyramidotomy suggest that a direct connectionbetween the primary motor cortex with the cervical spinal cord isessential for the recovery of voluntary skilled forelimb movement.

Example 13 Cortical Re-Organization Requires Rehabilitative Training

The repeated testing of skilled forelimb reach over the course of theexperiment essentially constitutes a rehabilitative training paradigmthat can promote motor recovery from spinal cord injury and corticalreorganization. In order to determine if the induced axonal plasticitymediated by Ryk deletion alone was required to promote functionalrecovery, the recovery of skilled forelimb reach was tested at 8 weeksafter CS injury in another cohort of mice that did not undergo weeklybehavioral testing after injury (FIG. 15A). Mice that did not undergoweekly behavioral testing displayed only limited skilled forelimbrecovery with performance similar to that of mice tested at one weekafter injury (FIGS. 15C and 8F). In the absence of weekly testing,refinement of cortical motor maps was also impaired, irrespective of Rykconditional deletion (FIGS. 15B and 22).

REFERENCES

-   1. Saxena, et al. (2007) Mechanisms of axon degeneration: from    development to disease. (Translated from eng) Progress in    neurobiology 83(3):174-191 (in eng).-   2. Wang, et al. (2012) Axon degeneration: molecular mechanisms of a    self-destruction pathway. (Translated from eng) The Journal of cell    biology 196(1):7-18 (in eng).-   3. Yan, et al. (2010) Axon degeneration: Mechanisms and implications    of a distinct program from cell death. (Translated from eng)    Neurochemistry international 56(4):529-534 (in eng).-   4. Schmidt, et al. (2009) Axon guidance proteins: novel therapeutic    targets for ALS? (Translated from eng) Prog Neurobiol 88(4):286-301    (in eng).-   5. Van Hoecke, et al. (EPHA4 is a disease modifier of amyotrophic    lateral sclerosis in animal models and in humans. (Translated from    eng) Nat Med 18(9):1418-1422 (in eng).-   6. Tury, et al. (Altered expression of atypical PKC and Ryk in the    spinal cord of a mouse model of amyotrophic lateral sclerosis.    (Translated from Eng) Dev Neurobiol (in Eng).-   7. Shi, et al. (2003) Hippocampal neuronal polarity specified by    spatially localized mPar3/mPar6 and PI 3-kinase activity.    (Translated from eng) Cell 112(1):63-75 (in eng).-   8. Nishimura, et al. (2004) Role of the PAR-3-KIF3 complex in the    establishment of neuronal polarity. (Translated from eng) Nature    cell biology 6(4):328-334 (in eng).-   9. Chen, et al. (2006) Microtubule affinity-regulating kinase 2    functions downstream of the PAR-3/PAR-6/atypical PKC complex in    regulating hippocampal neuronal polarity. (Translated from eng)    Proceedings of the National Academy of Sciences of the United States    of America 103(22):8534-8539 (in eng).-   10. Parker, et al. (2013) Competing molecular interactions of aPKC    isoforms regulate neuronal polarity. (Translated from eng)    Proceedings of the National Academy of Sciences of the United States    of America 110(35):14450-14455 (in eng).-   11. Zhang, et al. (2007) Dishevelled promotes axon differentiation    by regulating atypical protein kinase C. (Translated from eng)    Nature cell biology 9(7):743-754 (in eng).-   12. Mori, et al. (2009) An essential role of the aPKC-Aurora A-NDEL1    pathway in neurite elongation by modulation of microtubule dynamics.    (Translated from eng) Nature cell biology 11(9):1057-1068 (in eng).-   13. Parker, et al. (Competing molecular interactions of aPKC    isoforms regulate neuronal polarity. (Translated from eng) Proc Natl    Acad Sci USA 110(35):14450-14455 (in eng).-   14. Wolf, et al. (2008) Phosphatidylinositol-3-kinase-atypical    protein kinase C signaling is required for Wnt attraction and    anterior-posterior axon guidance. (Translated from eng) The Journal    of neuroscience: the official journal of the Society for    Neuroscience 28(13):3456-3467 (in eng).-   15. Onishi, et al. (Antagonistic Functions of Dishevelleds Regulate    Frizzled3 Endocytosis via Filopodia Tips in Wnt-Mediated Growth Cone    Guidance. (Translated from eng) J Neurosci 33(49):19071-19085 (in    eng).-   16. Wang, et al. (1999) Atypical PKC zeta is activated by ceramide,    resulting in coactivation of NF-kappaB/JNK kinase and cell survival.    (Translated from eng) Journal of neuroscience research 55(3):293-302    (in eng).-   17. Wooten, et al. (1999) Overexpression of atypical PKC in PC12    cells enhances NGF-responsiveness and survival through an NFkappaB    dependent pathway. (Translated from eng) Cell death and    differentiation 6(8):753-764 (in eng).-   18. Xie, et al. (2000) Protein kinase C iota protects neural cells    against apoptosis induced by amyloid beta-peptide. (Translated from    eng) Brain research. Molecular brain research 82(1-2):107-113 (in    eng).-   19. Huang, et al. (2001) Activation of protein kinase A and atypical    protein kinase C by A(2A) adenosine receptors antagonizes apoptosis    due to serum deprivation in PC12 cells. (Translated from eng) The    Journal of biological chemistry 276(17):13838-13846 (in eng).-   20. Kim, et al. (2007) Polarity proteins PAR6 and aPKC regulate cell    death through GSK-3beta in 3D epithelial morphogenesis. (Translated    from eng) Journal of cell science 120(Pt 14):2309-2317 (in eng).-   21. Joung, et al. (2005) p62 modulates Akt activity via association    with PKCzeta in neuronal survival and differentiation. (Translated    from eng) Biochemical and biophysical research communications    334(2):654-660 (in eng).-   22. Xin, et al. (2007) Protein kinase Czeta abrogates the    proapoptotic function of Bax through phosphorylation. (Translated    from eng) The Journal of biological chemistry 282(29):21268-21277    (in eng).-   23. Reyland M E (2009) Protein kinase C isoforms: Multi-functional    regulators of cell life and death. (Translated from eng) Front    Biosci (Landmark Ed) 14:2386-2399 (in eng).-   24. Liu, et al. (2005) Ryk-mediated Wnt repulsion regulates    posterior-directed growth of corticospinal tract. (Translated from    eng) Nature neuroscience 8(9):1151-1159 (in eng).-   25. Schmitt, et al. (2006) Wnt-Ryk signalling mediates    medial-lateral retinotectal topographic mapping. (Translated from    eng) Nature 439(7072):31-37 (in eng).-   26. Keeble, et al. (2006) Ryk: a novel Wnt receptor regulating axon    pathfinding. (Translated from eng) The international journal of    biochemistry & cell biology 38(12):2011-2017 (in eng).-   27. Keeble, et al. (2006) The Wnt receptor Ryk is required for    Wnt5a-mediated axon guidance on the contralateral side of the corpus    callosum. (Translated from eng) The Journal of neuroscience: the    official journal of the Society for Neuroscience 26(21):5840-5848    (in eng).-   28. Gonzalez, et al. (2013) The ryk receptor is expressed in glial    and fibronectin-expressing cells after spinal cord injury.    (Translated from eng) Journal of neurotrauma 30(10):806-817 (in    eng).-   29. Hollis E R, 2nd & Zou Y (2012) Reinduced Wnt signaling limits    regenerative potential of sensory axons in the spinal cord following    conditioning lesion. (Translated from eng) Proceedings of the    National Academy of Sciences of the United States of America    109(36):14663-14668 (in eng).-   30. Hollis E R, 2nd & Zou Y (2012) Expression of the Wnt signaling    system in central nervous system axon guidance and regeneration.    (Translated from eng) Frontiers in molecular neuroscience 5:5 (in    eng).-   31. Fradkin, et al. (2010) Ryks: new partners for Wnts in the    developing and regenerating nervous system. (Translated from eng)    Trends in neurosciences 33(2):84-92 (in eng).-   32. Liu, et al. (2008) Repulsive Wnt signaling inhibits axon    regeneration after CNS injury. (Translated from eng) The Journal of    neuroscience: the official journal of the Society for Neuroscience    28(33):8376-8382 (in eng).-   33. Miyashita, et al. (2009) Wnt-Ryk signaling mediates axon growth    inhibition and limits functional recovery after spinal cord injury.    (Translated from eng) Journal of neurotrauma 26(7):955-964 (in eng).-   34. Guenther, et al. (2012) Increased atypical PKC expression and    activity in the phrenic motor nucleus following cervical spinal    injury. (Translated from eng) Experimental neurology 234(2):513-520    (in eng).-   35. Luo L & O'Leary D D (2005) Axon retraction and degeneration in    development and disease. (Translated from eng) Annual review of    neuroscience 28:127-156 (in eng).-   36. Watts, et al. (2003) Axon pruning during Drosophila    metamorphosis: evidence for local degeneration and requirement of    the ubiquitin-proteasome system. (Translated from eng) Neuron    38(6):871-885 (in eng).-   37. Zhai, et al. (2003) Involvement of the ubiquitin-proteasome    system in the early stages of wallerian degeneration. (Translated    from eng) Neuron 39(2):217-225 (in eng).-   38. Xiao, et al. (2006) Insights into the mechanism of microtubule    stabilization by Taxol. (Translated from eng) Proceedings of the    National Academy of Sciences of the United States of America    103(27):10166-10173 (in eng).-   39. Drewes, et al. (1997) MARK, a novel family of protein kinases    that phosphorylate microtubule-associated proteins and trigger    microtubule disruption. (Translated from eng) Cell 89(2):297-308 (in    eng).-   40. Hurov, et al. (2004) Atypical PKC phosphorylates PAR-1 kinases    to regulate localization and activity. (Translated from eng) Current    biology: CB 14(8):736-741 (in eng).-   41. Matenia D & Mandelkow E M (2009) The tau of MARK: a polarized    view of the cytoskeleton. (Translated from eng) Trends in    biochemical sciences 34(7):332-342 (in eng).-   42. Shen H M & Liu Z G (2006) JNK signaling pathway is a key    modulator in cell death mediated by reactive oxygen and nitrogen    species. (Translated from eng) Free radical biology & medicine    40(6):928-939 (in eng).-   43. Manning A M & Davis R J (2003) Targeting JNK for therapeutic    benefit: from junk to gold? (Translated from eng) Nature reviews.    Drug discovery 2(7):554-565 (in eng).-   44. Yoshimura K, et al. (2011) c-Jun N-terminal kinase induces    axonal degeneration and limits motor recovery after spinal cord    injury in mice. (Translated from eng) Neuroscience research    71(3):266-277 (in eng).-   45. Li, et al. (2004) JNK-dependent phosphorylation of c-Jun on    serine 63 mediates nitric oxide-induced apoptosis of neuroblastoma    cells. (Translated from eng) The Journal of biological chemistry    279(6):4058-4065 (in eng).-   46. Onishi K, et al. (2013) Antagonistic Functions of Dishevelleds    Regulate Frizzled3 Endocytosis via Filopodia Tips in Wnt-Mediated    Growth Cone Guidance. (Translated from eng) The Journal of    neuroscience: the official journal of the Society for Neuroscience    33(49):19071-19085 (in eng).-   47. Tury, et al. (2013) Altered expression of atypical PKC and Ryk    in the spinal cord of a mouse model of amyotrophic lateral    sclerosis. (Translated from Eng) Developmental neurobiology (in    Eng).-   48. Kamitori. Et al. (1999) Expression of receptor tyrosine kinase    RYK in developing rat central nervous system. (Translated from eng)    Brain research. Developmental brain research 114(1):149-160 (in    eng).-   49. Lyu, et al. (2008) Cleavage of the Wnt receptor Ryk regulates    neuronal differentiation during cortical neurogenesis. (Translated    from eng) Developmental cell 15(5):773-780 (in eng).-   50. Halford M M, et al. (2000) Ryk-deficient mice exhibit    craniofacial defects associated with perturbed Eph receptor    crosstalk. (Translated from eng) Nature genetics 25(4):414-418 (in    eng).-   51. Wang, et al. (2002) Frizzled-3 is required for the development    of major fiber tracts in the rostral CNS. (Translated from eng) The    Journal of neuroscience: the official journal of the Society for    Neuroscience 22(19):8563-8573 (in eng).-   52. Hua, et al. (Frizzled3 controls axonal development in distinct    populations of cranial and spinal motor neurons. (Translated from    eng) Elife 2(0):e01482 (in eng).-   53. Shafer, et al. (Vangl2 promotes Wnt/planar cell polarity-like    signaling by antagonizing Dvl1-mediated feedback inhibition in    growth cone guidance. (Translated from eng) Dev Cell 20(2):177-191    (in eng).-   54. Macheda M L, et al. (The Wnt Receptor Ryk Plays a Role in    Mammalian Planar Cell Polarity Signaling. (Translated from Eng) J    Biol Chem (in Eng).-   55. Andre P, et al. (The Wnt coreceptor Ryk regulates Wnt/planar    cell polarity by modulating the degradation of the core planar cell    polarity component Vangl2. (Translated from Eng) J Biol Chem (in    Eng).-   56. Romanelli, et al. (1999) p70 S6 kinase is regulated by protein    kinase Czeta and participates in a phosphoinositide    3-kinase-regulated signalling complex. (Translated from eng)    Molecular and cellular biology 19(4):2921-2928 (in eng).-   57. Mairet-Coello G, et al. (2013) The CAMKK2-AMPK kinase pathway    mediates the synaptotoxic effects of Abeta oligomers through Tau    phosphorylation. (Translated from eng) Neuron 78(1):94-108 (in eng).

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. An isolated anti-Ryk antibody or antibody fragment that: (a)specifically binds to a binding domain of Wnt, the antibody or antibodyfragment comprising a heavy chain variable region comprising the CDRsequences set forth in SEQ ID NOs: 5-7; and/or a light chain variableregion comprising the CDR sequences set forth in SEQ ID NOs: 1-3; or (b)specifically binds to the same epitope on Wnt as does a referenceantibody or antibody fragment, or cross-competes for specific binding toWnt with a reference antibody or antibody fragment, said referenceantibody or antibody fragment comprising a heavy chain variable regioncomprising the CDR sequences set forth in SEQ ID NOs: 5-7; and/or alight chain variable region comprising the CDR sequences set forth inSEQ ID NOs: 1-3.
 2. The isolated anti-Ryk antibody or antibody fragmentof claim 1, wherein the antibody or antibody fragment inhibits orreduces Ryk binding to Wnt.
 3. The isolated anti-Ryk antibody orantibody fragment of claim 1, wherein the antibody or antibody fragmentspecifically binds to an epitope within amino acid residues 90-183 ofWnt.
 4. The isolated antibody or antibody fragment of claim 1, whereinthe heavy chain variable region comprises an amino acid sequencecomprising at least 85% sequence identity to SEQ ID NO: 8, optionally atleast 90% sequence identity to SEQ ID NO:
 8. 5. The isolated antibody orantibody fragment of claim 1, wherein the heavy chain variable regioncomprises an amino acid sequence comprising at least 95% sequenceidentity to SEQ ID NO: 8, optionally at least 99% sequence identity toSEQ ID NO:
 8. 6. The isolated antibody or antibody fragment of claim 1,wherein the heavy chain variable region comprises the amino acidsequence set forth in SEQ ID NO:
 8. 7-9. (canceled)
 10. The isolatedantibody or antibody fragment of claim 1, wherein the light chainvariable region comprises an amino acid sequence comprising at least 85%sequence identity to SEQ ID NO: 4, optionally at least 90% sequenceidentity to SEQ ID NO:
 4. 11. The isolated antibody or antibody fragmentof claim 1, wherein the light chain variable region comprises an aminoacid sequence comprising at least 95% sequence identity to SEQ ID NO: 4,optionally at least 99% sequence identity to SEQ ID NO:
 4. 12. Theisolated antibody or antibody fragment of claim 1, wherein the lightchain variable region comprises the amino acid sequence set forth in SEQID NO:
 4. 13-15. (canceled)
 16. An isolated antibody or antibodyfragment that specifically binds to a binding domain of Wnt, comprising:a) a heavy chain variable region comprising at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 8, the heavychain variable region comprising three CDR sequences set forth in SEQ IDNOs: 5-7; and/or b) a light chain region variable region comprising atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO: 4, the light chain variable region comprising three CDR sequencesset forth in SEQ ID NOs: 1-3.
 17. The isolated antibody or antibodyfragment of claim 16, wherein the heavy chain variable region comprisesat least 90% sequence identity to the amino acid sequence of SEQ ID NO:8 and the light chain variable region comprises at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO:
 4. 18. The isolatedantibody or antibody fragment of claim 16, wherein the heavy chainvariable region comprises at least 95% sequence identity to the aminoacid sequence of SEQ ID NO: 8 and the light chain variable regioncomprises at least 95% sequence identity to the amino acid sequence ofSEQ ID NO:
 4. 19. The isolated antibody or antibody fragment of claim16, wherein the heavy chain variable region comprises at least 99%sequence identity to the amino acid sequence of SEQ ID NO: 8 and thelight chain variable region comprises at least 99% sequence identity tothe amino acid sequence of SEQ ID NO:
 4. 20. The isolated antibody orantibody fragment of claim 16, wherein the heavy chain variable regioncomprises the amino acid sequence of SEQ ID NO: 8 and the light chainvariable region comprises the amino acid sequence of SEQ ID NO: 4.21-25. (canceled)
 26. A pharmaceutical composition comprising aneffective amount of the antibody or antibody fragment according to claim1, and a pharmaceutically acceptable carrier or excipient.
 27. A nucleicacid sequence encoding the isolated antibody or antibody fragmentaccording to claim
 1. 28. A vector comprising the nucleic acid sequenceof claim
 27. 29. The vector of claim 28, wherein the vector is anexpression vector.
 30. A host cell comprising the vector of claim 28.31. The host cell of claim 30, wherein the host cell is a mammalian hostcell.
 32. A transgenic mouse comprising the host cell of claim 30,wherein the mouse expresses a polypeptide encoded by the nucleic acid.33-61. (canceled)
 62. An immunoconjugate comprising the isolatedanti-Ryk antibody or antibody fragment of claim 1, linked to atherapeutic agent.
 63. The immunoconjugate of claim 62, wherein thetherapeutic agent is a cytotoxin or a radioactive isotope.
 64. Abispecific molecule comprising the isolated anti-Ryk antibody orantibody fragment of claim 1, linked to a second functional moietyhaving a different binding specificity than the isolated anti-Rykantibody or antibody fragment of claim
 1. 65-75. (canceled)
 76. Apharmaceutical composition comprising an effective amount of theantibody or antibody fragment according to claim 16, and apharmaceutically acceptable carrier or excipient.
 77. A nucleic acidsequence encoding the isolated antibody or antibody fragment accordingto claim
 16. 78. A vector comprising the nucleic acid sequence of claim77.
 79. A host cell comprising the vector of claim
 78. 80. A transgenicmouse comprising the host cell of claim 79, wherein the mouse expressesa polypeptide encoded by the nucleic acid.
 81. An immunoconjugatecomprising the isolated anti-Ryk antibody or antibody fragment of claim16, linked to a therapeutic agent.
 82. The immunoconjugate of claim 81,wherein the therapeutic agent is a cytotoxin or a radioactive isotope.83. A bispecific molecule comprising the isolated anti-Ryk antibody orantibody fragment of claim 16, linked to a second functional moietyhaving a different binding specificity than the isolated anti-Rykantibody or antibody fragment of claim 16.